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SNPlex Genotyping System 48-plex Automating OLA Using the

1. Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Creating and Naming the Deck Layout Creating and Naming the Deck Layout Building the deck layout includes both physically building the deck on the instrument and modelling the physical deck in the Biomek FX software Deck Editor In the Deck Editor name the deck layout file ABI SNPLEX NC IMPORTANT Build the deck layout before the Installation and Operation Qualifications IQ OQ are performed The following illustration shows the positions on an empty deck for the Biomek FX TL1 Tip Loader P1 to p16 16 position ALP 4 x 4 P17 to P18 2 single position ALPs In the figure above e Positions P1 through P16 comprise a single 4X4 hardware component the 16 position ALP Positions P17 and P18 are individual 1X1 single position ALPs e The tip loader ALP is at TL 1 Note If you have a new Biomek FX instrument a Beckman Coulter representative must perform IQ OQ using the physical deck layout and virtual deck layout that you just built 12 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Framing the Deck Framing the Deck Before you can use the Biomek FX instrument you must calibrate the positions on the deck Biomek FX documentation refers to this process as framing the deck D
2. Performing OLA s 3 Performing OLA V Using Dry DNA 4 NSZ Using Wet DNA See page 64 Overview Purifying OLA Exonuclease Preparing the Reagents Diluting the Purified A OLA Product Running Biomek FX See page 66 Method 4 PCR Setting Up the PCR Reactions Notes SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 63 commas Chapter 7 Setting Up the PCR Reactions ES Overview Overview About this This chapter provides information on how to prepare four SNPlex PCR reaction plates Chapter from four SNPlex OLA reaction plates using the Biomek FX liquid handling instrument Assumptions This protocol assumes that four 384 well SNPlex OLA reaction plates are available and the contents previously exonuclease purified and diluted One Biomek FX method is available for assembling the SNPlex system based PCR reactions Where You Are In the SN Plex Design and order System Assay SNPlex ligation probes Workflow Design sample plate layout Run PCR Prepare hybridization plates OLA Laboratory PCR Laboratory Prepare gDNA and bind PCR product to plates z Ke DA s E Phosphorylate and Phosphorylate and PUNI ib aoi ied 2 ligate probes to ligate primers to Og SC AT cine N e hybridization plate gDNA OLA dry gDNA OLA wet So D gt T Hybridize ZipChute probes 2 Purify O
3. 4 When the method pauses verify the deck layout and click OK to continue 5 When the method pauses again remove and discard the eight used tip boxes 56 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 5 Purifying OLA Products Exonuclease Running Biomek FX Method 2 Exo EE 6 Remove the tips corresponding to well positions G12 and H12 from eight new uncovered tip boxes and place them on the deck as shown in the deck layout below Plate Position Deck Layout 1 OLA1 to OLAA P5 to P8 Uncovered tip P9 to P16 box Exo Mix F18 f Click OK to continue the method 8 After the method completes remove all eight used tip boxes and the Exo Mix plate 9 Remove seal and briefly centrifuge the OLA reaction plates OLAI to OLA4 10 Proceed to Thermal Cycling the OLA Reactions Thermal Cycling Thermal cycle the OLA plates using the following conditions the OLA Reactions Step Step Type Temperature C Time 1 HOLD 37 90 min 2 HOLD 80 10 min 3 HOLD 4 co Next Steps Atthis point the OLA purification reaction is complete Proceed to Chapter 6 Diluting the Purified OLA Product SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 57 Chapter 5 Purifying OLA Products Exonuclease Running Biomek FX Method 2 Exo 58 SNPlex Genotyping Sys
4. Label four 384 well ABI PRISM clear optical reaction plates OLA1 to OLA4 For information on the layout of wet gDNA plates see Method ld wet on page 37 Remove the tips corresponding to well positions G12 and H12 from five tip boxes Qs DOO OF OGO0GOOOd 60000000 00000000 0p 00000 oo o000q 600009 00000000 9 0 6 9 9 0 0 Q OOOOOOG Remove tips from wells G12 and H12 Place the five uncovered tip boxes and the plates on the deck as shown in the deck layout below Plate Position Method 1d_wet Deck Layout 1 OLA1 to OLAA P1 to PA Assay Mix P18 DNA1 to DNA4 P5 to P8 Uncovered tip P9to P12 box P17 Start the method using ld SNPLEXv5 OLA P1 S16 W bmt 50 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Method 1d wet 4 When the method pauses click OK to continue 5 When the method pauses remove and discard the four used tip boxes from P9 to P12 6 Seal and store if necessary the DNA sample plates DNA1 to DNA4 from positions P5 to P8 f Remove the tips corresponding to well positions G12 and H12 from four new uncovered tip boxes and place them and the plates on the deck as shown in the deck layout below Plate Position Method 1d wet Deck Layout 2 OLA1 to OLAA P1 to
5. P1 OLA1 EE E E 5 Click OK to continue the method 6 When the method pauses remove the tips corresponding to well positions G12 and H12 from eight new uncovered tip boxes and replace the used tip boxes in positions P9 to P16 f Click OK to continue the method 8 After the method completes remove seal and store if necessary the DNA sample plates DNA1 to DNA4 9 Remove seal and briefly centrifuge the OLA reaction plates OLA1 to OLA4 10 Proceed to Thermal Cycling the OLA Reactions on page 44 SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 43 Chapter 4 Performing OLA Using Wet gDNA e Performing the OLA Reactions Biomek FX Methods 1a wet or 1b wet Thermal Cycling Thermal cycle the OLA plates using the following conditions the OLA Heactions Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 cycles 94 15 sec 60 30 sec 51 396 ramp 30 sec 4 HOLD 99 10 min 5 HOLD 4 M Next Steps At this point the OLA reaction is complete Proceed to Chapter 5 Purifying OLA Products Exonuclease 44 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Method 1c_wet Performing the OLA Reactions Biomek FX Method 1c_wet About the Method Meth
6. Plate Position Method 1c dry Deck Layout 3 OLA1 to OLAA P1 to PA i igg HH rrr woetoMki2 rstore MAR LE ES Uncovered tip P9 to P12 Mix10 H af Mix11 i 7 oe Mix12 J ill L 8 Click OK to continue The Biomek FX instrument transfers OLA mix from plates Mix9 to Mix12 to plate OLA3 and then pauses 30 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Thermal Cycling the OLA Reactions Next Steps Chapter 3 Performing OLA Using Dry gDNA EG c Performing the OLA Reactions Biomek FX Method 1c ary QA 9 Remove the used tip boxes and plates Mix9 to Mix12 then replace the tip boxes and plates leaving the OLA reaction plates in place as specified in the following table Plate Position Method 1c dry Deck Layout 4 OLA1 to OLA4 P1 to P4 n to P5 to P8 N Uncovered tip P9 to P12 box Pl P13 P15 P16 10 Click OK to continue The Biomek FX instrument transfers OLA mix from plates Mix13 to Mix16 to plate OLA4 11 After the method run is complete remove and discard the tip boxes and plates Mix13 to Mix16 and remove seal and briefly centrifuge the OLA reaction plates OLAI to OLA4 12 Proceed to Thermal Cycling the OLA Reactions Thermal cycle the OLA plates using the following conditions Step Step Type Temperature C Time 1 HOLD 4
7. 4 positions of a 96 well quadrant are reserved for 1 control DNA 1 NTC and 2 Probe Pool S allelic ladder wells Total number of samples in a batch containing four 384 well plates T Method numbering reflects methods provided for other SNPlex System assay protocols tP 23 SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Methods 1a dry or 1b dry Performing the OLA Reactions Biomek FX Methods 1a dry or 1b dry About These Methods la dry and 1b dry were developed for setups that have 368 gDNA samples Methods and four probe pools These methods use a single identical deck layout The difference between them is the pattern in which the instrument dispenses the reagents into the wells of the OLA reaction plates In Method la dry Assay Mix from source plate Mix is transferred to all four quadrants of OLA reaction plate OLA1 Mix 2 to OLA2 Mix 3 to OLA3 and Mix 4 to OLA4 In Method 1b dry Assay Mix from source plate Mix 1 is transferred to quadrant 1 of all four OLA reaction plates OLA1 to OLA4 Mix 2 to quadrant 2 Mix 3 to quadrant 3 and Mix 4 to quadrant 4 Preparing the 1 Thaw the SNPlex Oligonucleotide Ligation Kit components and probe pools at Reagents room temperature 2 Label four skirted 96 well PCR plates Mix to Mix4 3 Label four 15 mL centrifuge tubes Mix to M
8. 0000000000000000 0000000000000000 OOOOOOOOOOOOOOOO0 eOO0O0OOOOOOOOOOOOO 000000000O0O0OOOOO OOOOOOOOOOOOOOOO time OOOOOOOOOOOOO oo0pooooooooooooo O0pooooooooooooo m QuwuOr 5xazzoa Method Numbert y 1b_dr 1c_dry SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 22 Chapter 3 Performing OLA Using Dry gDNA Plate Layouts and Methods for the OLA Protocol Dry gDNA Reference Performing the OLA Reactions Biomek FX Method 1d dry on page 32 T TII amp OOOOOOOO 8OOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOO0O00 amp OOOOOOOOOOOOOO0O00 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO cOOOOOOOOOOOOOOO0 0OO0OOOOOOOOOOOOOO0 000000O0O0OOOOOOOO O0O0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO OO0OOOOOOOOOOOOOO 000 0000000000000 2000000000000000 s o0O000000000000000 ooooo0oo00000000000 Allelic Ladder lt Z O o c o Q aoooooo0oo0000000000 amp OOOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO TOOOOOOOOOOOOOOOO 000O0OOOOOOOOOOOO 0000000O0O0OOOOOOO 0O0O0OOOOOOOOOOOOO0 00O0OOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 eOOOOOOOOOOOOOOOO 000000000O0OOOOOO OOOOOOOOOOOOOOOO eOO0OOOOOOOOO
9. Appendix A of the SNPlex Genotyping System 48 plex User Guide Item Vendor io SNPlex System Kits Required for This Protocol SNPlex System Oligonucleotide Ligation Kit Applied Biosystems 4357460 e SNPlex OLA Master Mix e SNPlex Universal Linkers 48 plex e SNPlex dATP 100X SNPlex System Ligation Probes Applied Biosystems 4346978 SNPlex System Purification Kit Applied Biosystems 4349357 e SNPlex Exonuclease Buffer 10X e SNPLex Lamda Exonuclease e SNPlex Exonuclease SNPlex System Amplification Kit Applied Biosystems 4349358 e SNPlex Amplification Master Mix 2X e SNPlex Amplification Primers 20X Other Reagents Nuclease free water Promega P119C SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 1 Introduction Required Equipment and Materials Item Vendor NI Sterile 1X TE buffer 10 mM Tris base pH 8 0 and 1 mM Fluka 93283 Na EDTA Documentation Document Name iun SNPlex Genotyping System 48 plex Assay Ordering Guide 4357460 SNPlex Genotyping System 48 plex User Guide 4360856 SNPlex Genotyping System 48 plex Quick Reference Card 4360855 SNPlex Genotyping System 48 plex General Automation Getting Started Guide 4363143 Biomek FX User s Manual Beckman Coulter 719452 SNPlex Genotyping System 48 plex Automating PCR Using the Tomtec 4
10. C o 9 Q1 29 9 4 59 s e9 73 Ge r8 m9 G94 Go G10 18 G28 Go 2 50 G58 Gee c 88 o9 1 19 2 G d ep s e 79 e3 c r Qs E God 6r G19 2 e Ed G5 Gad e D 8e o3 13 Qe G28 a9 4 Qs e 79 Ge D e e Ce God 12 o G28 Go 9 652 Qe Ge E CD o9 19 Qe 9 49 amp de 99 M E Get Gad Qs Gos G13 Ge G2 GD 49 69 66 F 98 o9 19 2 o9 Gd 49 Qs 6 79 79 t F Ged e e Gos 19 e Ge Gd Gao 9 662 CN e 89 o 19 Qe Gs 29 4 s e9 7 69 c Gas Go os Gor rs Gej Get 6 9 64 Gd Gos H C o9 19 e C 49 49 59 e 673 Ged H Ge Ge Go Gos Gre e 693 G40 Gu 58 66 Q9 Volumes for Source Plates The volumes of the gDNA samples you use depend on your total number of samples and the number of SNPlex Ligation Probe pools you are using The volumes in the table below include dead volumes to be sure the robotic tips remain submerged during aspiration Method Number of Number of Number of Volume as Number MODO Siemans Probe Pools SPNA Source 9DNA of gDNA amples Samples 1a wet 1a SNPLEXv5 OLA PN S4 W bmt 4 4 368 20 1b wet 1b SNPLEXv5 OLA P4 SN W bmt 368 20 1c wet 1c SNPLEXv5 OLA P16 S1 W bmt 16 1 92 40 1d wet 1d SNPLEXv5 OLA P1 S16 W bmt 1 16 1472 12 SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 37 Plate Layouts and Methods for the O
11. H12 from nine tip boxes Method 1 o 1 00 90 OQ OG 6000000000009 6000000000000 0 0 0 0 0 0 0 Q 99000000 900000000 9000000 OQOOOOOG OOOQOOOOGY e e 0000 6660 600000000 p 00 00 Remove tips from wells G12 and H12 2 Place an ACME adapter underneath each PCR plate then place the nine uncovered tip boxes and the plates on the deck as shown in the deck layout below Plate Position Deck Layout 1 PCR1 to PCR4 P1 to P4 OLA1 to OLAA P5 to P8 PCR Mix P18 Uncovered tip P17 P9 to box P16 3 Start the method using 2 SNPLEXv5 PCR bmt 4 When the method pauses click OK to continue 5 When the method next pauses remove and discard the eight used tip boxes 66 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 7 Setting Up the PCR Reactions Running Biomek FX Method 4 PCR me 6 Remove the tips corresponding to well positions G12 and H12 from eight new uncovered tip boxes and place them on the deck as shown in the deck layout below Plate Position Deck Layout 2 PCR1 to PCR4 P1 to P4 OLA1 to OLAA P5 to P8 PCR Mix P18 Uncovered tip P17 P9 to box P16 PCR_Mix F18 f Click OK to continue the method 8 After the method completes remove the used tip boxes and discard the PCR Mix plate 9 Remove sea
12. Neat HAZARDOUS WASTE Refer to Material Safety Data Sheets Hazard MSDSs and local regulations for handling and disposal Chemical Waste To minimize the hazards of chemical waste Safety Guidelines SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Read and understand the Material Safety Data Sheets MSDSs provided by the manufacturers of the chemicals in the waste container before you store handle or dispose of chemical waste Provide primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Handle chemical wastes in a fume hood After emptying the waste container seal it with the cap provided Dispose of the contents of the waste tray and waste bottle in accordance with good laboratory practices and local state provincial or national environmental a
13. O O COCO 600009 0000 90000000000 60000000000 900 0 0 0 0 0 OOOOOOO 9900000 0 0 0 0 0 9 00000009 90000 ee 0000 Remove tips from wells G12 and H12 2 Place the uncovered tip boxes and plates on the deck as shown in the following figure placing an ACME adapter underneath each OLA plate Plate Position Method 1d dry Deck Layout 1 Uncoveredtip TL1 box AssayMix P18 OLA1 to OLAA P1 to P4 ix 3 Start the method using Id SNPLEXv5 OLA P1 S16 D bmt 4 When the method pauses verify that the deck layout corresponds to the deck layout above and click OK to continue N lo tes INOTES SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 33 Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Method 1d dry 5 After the method run is complete remove and discard the tip boxes and the Assay Mix plate and remove seal and briefly centrifuge the OLA reaction plates OLA1 to OLA4 6 Proceed to Thermal Cycling the OLA Reactions Thermal Cycling Thermal cycle the OLA plates using the following conditions the OLA Reactions Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 cycles 94 15 sec 60 30 sec 51 396 ramp 30 sec 4 HOLD 99 10 min 5 HOLD 4 M Next Steps At this point
14. SN D bmt Plate 3 Plate 1 oooooooooooool OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO TOOOOOOOOOOOOOOOO 0000O0OOOOOOOOOOO 000O0OOOOOOOOOOOO SO0O0OOOOOOOOOOOOOO O0O0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 eO0O0O0OOOOOOOOOOOOO 000000000O0OOOOOO OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO so0o000000000000000 vOOOOOOOOOOOOOOOO time OOOOOOOOOOOOO 20 21 22 23 24 OOOOO OOOOCO o60pooooooooooooo o06lpooooooooooooo m QuwuOr 5xazzon amp OOOOOOOO eo o amp OOOOOOOO jJO 0 0 9j0 e e e 8OOOOOOOOOOOOOOO0 amp 0000000000000000l amp OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 0000000000000000 zooooo0oo00000000000 00OOOOOOOOOOOOO0 zooo0o0000000000000 z zooo0oo0000000000000 zoooo0o000000000000 ooooo0oo00000000000 OOOOOOOOOOOOOOOO 0000000000000000 20000000000000000 00000000000 00000 o0oo0o00000000000000 eo000000000000000 s o0O000000000000000 OOOOOOOOOOOOOOOO time OOOOOOOOOOOOO x DOOOOOOOOOOOOO DOOOOOOOOOOOOO m QuuwuOr 5xazzon Plate 2 Control DNA Allelic Ladder NTC 8OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO0 0OOOOOOOOOOOOOOOO 0O0OOOOOOOOOOOOOO TOOOOOOOOOOOOOOOO0 00000O0O0OOOOOOOOO 0000OOOOOOOOOOOO zZOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 eOOOOOOOOOOOOOOOO 0000000000O0OOOO
15. and plates on the deck as shown in the following figure placing an ACME adapter underneath each OLA plate Plate Position Method 1c dry Deck Layout 1 OLA1 to OLAA P1 to PA Mix1 to Mix4 P5 to P8 Uncovered tip P9 to P12 box AJ L2 OTAQ E LV ENV SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 29 Chapter 3 Performing OLA Using Dry gDNA CMS Performing the OLA Reactions Biomek FX Method 1c ary 3 Start the method using lc SNPLEXv5 OLA P16 SI D bmt 4 When the method pauses verify that the deck layout corresponds to the deck layout above and click OK to continue The Biomek FX instrument transfers OLA mix from plates Mix to Mix4 to plate OLA1 and then pauses 5 Remove the used tip boxes and plates Mix1 to Mix4 then replace the tip boxes and plates leaving the OLA reaction plates in place as specified in the following table Plate Position Method 1c dry Deck Layout 2 OLA1 to OLAA P1 to PA Mix5 Mix5 to Mix8 P5 to P8 Tu P13 Uncovered tip P9 to P12 mR Mix6 HH box HE Mix 33 m LE Mix8 i OE 6 Click OK to continue The Biomek FX instrument transfers OLA mix from plates Mix5 to Mix8 to plate OLA2 and then pauses f Remove the used tip boxes and plates Mix5 to Mix8 then replace the tip boxes and plates leaving the OLA reaction plates in place as specified in the following table
16. bulk mixture into 96 well source plate Use the volumes in this column to prepare the reagents Refer to the SNPIex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Running 1 Method 1c wet Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Method 1c wet Add 13 uL of the Assay Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells Mix n l00000000000 090000000000 13 uL 900000000000 000000000000 00000000000 Y 00000000000 90000000000C 000000000000 Leave wells G12 and H12 empty Label four 384 well clear optical reaction plates OLA1 to OLA4 For information on the layout of wet gDNA plates see Method 1c wet on page 37 Remove the tips corresponding to well positions G12 and H12 from every tip box Qs 000b a OCOO000006 6600000000 q 6 000 00000 0Q OOOOOOOG OOOO OOOOOOOE OQOOOOOOG Oo 000 000000 Remove tips f
17. dry gDNA OLA wet oS Hybridization Reagents and Kits Hybridize ZipChute probes Purify OLA products Exonuclease Purification Kit Elute ZipChute probes Dilute purified OLA product Assay Standards Kit Prepare sample plates for electrophoresis Prepare PCR reactions Amplification Kit Create results groups and plate records Load and run sample plates Analyze data in GeneMapper software v3 7 60 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 6 Diluting the Purified OLA Product Running Biomek FX Method 3 Dilution gt Running Biomek FX Method 3 Dilution About this This method uses two deck layouts The first deck layout adds water to OLA reaction Method plates OLAI and OLA2 The second deck layout adds water to OLA reaction plates OLA3 and OLA4 The well contents are mixed each time after the water is dispensed Preparing the Add 50 mL of nuclease free water to an Axygen reservoir Reagent Running This 1 Remove the tips corresponding to well positions G12 and H12 from eight tip boxes Method Remove tips from wells G12 and H12 2 Place the eight uncovered tip boxes the water reservoir and plates on the deck as shown in the deck layout below Plate Position Deck Layout 1 OLA1 to OLA4 P5 to P8 Water P18 Uncovered tip P9 to P16 box 3 St
18. to P12 box NA15 NAT L Mix j Assay Mix P18 EE ES EH Click OK to continue the method After the method run is complete remove and discard the tip boxes Seal and store plates DNA13 to DNA16 if necessary Remove the Assay Mix plate and discard Remove seal and briefly centrifuge the OLA reaction plates OLA1 to OLA4 Proceed to Thermal Cycling the OLA Reactions Thermal Cycling Thermal cycle the OLA plates using the following conditions the OLA Reactions Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 cycles 94 15 sec 60 30 sec 51 396 ramp 30 sec 4 HOLD 99 10 min 5 HOLD 4 so Next Steps At this point the OLA reaction is complete Proceed to Chapter 5 Purifying OLA 52 Products Exonuclease SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide J Introduction Setting Up the _ Biomek FX for Automating the SNPlex System Assay Performing OLA s Performing OLA Dor Ec Using Dry DNA v ims Using Wet DNA Purifying OLA iiemaspvou wuss Products XT r Exonuclease c Diluting the Purified 775777 77 OLA Product Setting Up the PCR Reactions Notes Chapter 5 Purifying OLA Products Exonuclease See page 54 Overview Running Biomek FX See page 5
19. 0 j ooo0o0000000000000 ka OCOO0O0000000000 CE E o E n Performing the OLA Reactions Method 1d_wet on page 49 seopooooo0oo00000000 O00pooooooooooooo m amp OOOOOOOO O Ojo eje ee e 8OOOOOOOOOOOOOOOO la0000000000000000 amp OOOOOOOOOOOOOOOO 00OOOOOOOOOOOOO0 0000OOOOOOOOOOOO OOOOOOOOOOOOOO0O00 00O0OOOOOOOOOOOOO 0000O0OOOOOOOOOOO 00O0OOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 eOOOOOOOOOOOOOOOO 000O0O0O0OOOOOOOOOO OOOOOOOOOOOOOOOO eOO0OOOOOOOOOOOOOO0 00000000000O0O0OOO OOooooooooooooooOo ga OOOOOOOOOOOOO o0poooooooooooooO O006pooooooooooooo m QuwuOr 5x azzon 1472 1d SNPLEXv5 OLA P1 S16 W bmt Plate 3 Plate 1 a SN eE OOOOOO0 OOOOOOOOOOOOOOO OOOOOOOOOOOOOO0 OOOOOOOOOOOOOO0O0 OOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 0000O0OOOOOOOOOOO S0O0O0O0O0OOOOOOOOOOO OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOO0 OO0O0OOOOOOOOOOOOO OOOOOOOOOOOOOOO0 eOOOOOOOOOOOOOOOO eOO0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 time OOCOOOOOOOOOOO 19 20 21 22 23 24 OOOOOO oo60pooooooooooooo O006poooooooooooooOo m QuwuOr 5x azzon EEE HHHH oo0oo00000 OOOOOOOOOOOOOOO OOOOOOOOOOOOOOO OOOOOOOOOOOOOO0O 000000000000000 ooooo00000000000 oooo0oo000000000
20. 00 OOOOOOOOOOOOOOCO OOOOOOOOOOOOOOO OOOOOOOOOOOOOO00 OOOOOOOOOOOOOO0O0 OOOOOOOOOOOOOOO OOOOOOOOOOOOOOCO O0000000000000000 eO0O0OOOOOOOOOOOOO0 0000000000000000 OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO dI OCOOOOOOOOOOOO 11 12 13 14 15 16 17 18 19 20 21 22 23 24 OOOOOOOOOOOOOO eopooooooooooooo 006pooooooooooooo m QuwuOr 5xazzomn Plate 4 Control DNA _ D O 9 G sai 2 D lt x 8OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 000O0OOOOOOOOOOOO 000O0OOOOOOOOOOOO 0O0OOOOOOOOOOOOOO O0OOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 eOO0OOOOOOOOOOOOOO 000000000O0OOOOOO OOOOOOOOOOOOOOOO eOO0OOOOOOOOOOOOOO OO0O0OOOOOOOOOOOOO0 vOOOOOOOOOOOOOOOO ga OOOOOOOOOOOOO o0poooooooooooooOo o00pooooooooooooo m QuwuOor 5x azzoa 8OOOOOOO000000000 lxooooooooooooooool 80000000000000000 0000000000000000 0000000000000000 0000000000000000 0000000000000000 20000000000000000 30000000000000000 20000000000000000 0000000000000000 0000000000000000 20000000000000000 20000000000000000 20000000000000000 0000000000000000 e0000000000000000 e0000000000000000 0000000000000000 da 0000000000000 oo0pooooooooooooo O06pooooooooooooo m Quucor 5xazzoma Method Number 1
21. 358100 Quadra 3 Getting Started Guide SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide T Chapter 1 Introduction Designing the Sample Plate Layout Designing the Sample Plate Layout Each 384 well plate is divided into four quadrants each with 96 wells The convention used in this document is that the first of 96 wells in quadrant 1 is well Al well B1 for quadrant 2 A2 for quadrant 3 and B2 for quadrant 4 12 p4 596 7 8 9 10 t1 12 13 14 15 16 17 18 19 20 21 22 23 24 M Ooo pooooooooooooooooooooo 15eobooooooooooooooooooooo Control DNA CPPPUV OOOOOOOODOOOOOO0O0O00O000 DOOOOOOOOOOOOOOOOOOOOOO000 EOOOOOOOOOOOOOOOOOOOOOOO0 NTC FOOOOOOOOOOOOOOOOOOOOOOO0 GOOOOOOOOOOOOOOOOOOOOOO000 HOOOOOOOOOOOOOOOOOOOOOO000 l a erred JoOo0o00000000000000000000 a ee LOOOOOOOOOOOOOOOOOOOOOO0 MOOOOOOOOOOOOOOOOOOO000 Allelic Ladder NOOOOOOOOOOOOOOOOOOO000 ooooooooooooooooooooooo POOOOOOOOOOOOOOOOOOOOOO0 A1 Quadrant 1 A2 Quadrant 3 B1 Quadrant2 B2 Quadrant 4 A number of plate layouts are possible assuming that each batch consists of four 384 well plates as illustrated in Plate Layouts and Methods for the OLA Protocol Dry gDNA on page 21 and Plate Layouts and Methods for the OLA Protocol Wet gDNA on page 37 The layout of a sample plate must be coordinated with the structure and naming of Data Collection software run folders in order for GeneMapper sof
22. 5 Method 2 Exo SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 53 E Chapter 5 Purifying OLA Products Exonuclease E E CCELI Overview Overview About This This chapter provides information on automating the addition of the Exonuclease mix to Chapter the OLA reactions using the Biomek FX Instrument The procedures in this chapter assume that you have completed the OLA preparation using either dry Performing OLA Using Dry gDNA on page 19 or wet Performing OLA Using Wet gDNA on page 35 gDNA Where You Are In the SN Plex Design and order System Assay SNPlex ligation probes Workflow Design sample plate layout D O Run PCR O 2 a o c o Prepare gDNA T Prepare hybridization plates o and bind PCR product Ss to plates g D G O 2 2 Phosphorylate and Phosphorylate and BS Add denaturant isolating 2 9 G ligate probes ligate probes 2 biotinylated strand on 2 linkers and linkers and as hybridization plate gDNA OLA dry gDNA OLA wet oS O l IS E gt am T Hybridize ZipChute probes 2 Purify OLA products E Exonuclease x z Elute ZipChute probes g Dilute purified OLA product S n G l Prepare sample plates 2 5 for electrophoresis w Prepare PCR reactions i o E lt x Create results groups and plate records Load and run sample plates Analyze data in GeneMap
23. 68 1b SNPLEXv5 OLA P4 SN W bmt Plate 3 Plate 1 s EEEH amp OOOOOOOO 0000000000000000 amp OOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO 00OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 TOOOOOOOOOOOOOOOO 0000O0OOOOOOOOOOO 0OO0O0OOOOOOOOOOOOO ZOOOOOOOOOOOOOOOO 000O0OOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO0 0000000000000O0O00 OOOOOOOOOOOOOOO00 eOO0O0OOOOOOOOOOOOO 20000000000000000 ooo0oo0o000000000000 tie OOO0000000000 eo0pooooooooooooo O00pooooooooooooo m QuwuOr 5x azzon Plate 4 Allelic Ladder lt Z QO 2 4 c o O EEEH amp OOOOOOOO 0000000000000000 z0000000000000000 amp OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 COOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 0OO0O0OOOOOOOOOOOO0 ZOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOO00 eOO0O0OOOOOOOOOOOO00 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 eOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 amp OOoooooooleooeoeeee amp Ooooooooocoejoocjleeeoe 8OOOOOOOOOOOOOOOCO amp OOOOOOOOOOOOOOOO SOOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOOCO OOOOOOOOOOOOOOOO TOOOOOOOOOOOOOOOO 000OOOOOOOOOOOOO O000O0O0OOOOOOOOOOO ZOOOOOOOOOOOOOOOO 20000O0OOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 270000000000000000 0000O0OOOOOOOOOOO OOOOOOOOOOOOOOOCO eOO0OOOOOOOOOOOOOO 2 000000O0O0OOOO
24. 700 0 94 tips in source plate x 10uL per tip Cr H Transfer losses for transferring bulk mixture into 96 well source plate Use the volumes in this column to prepare the reagents Refer to the SNPIex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide 32 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Method 1d ary 4 Add 90 uL of the Assay Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells o ES S 000080000 0008080000 Oe TIT TT Mix 1 T 90 uL eoc000000 ALLALI J 000000080 ecc00000 ec000000 NS Leave wells G12 and L N D o 3 lt 5 Label four 384 well plates containing dried DNA OLA1 to OLA4 For information on the layout of dry gDNA plates see Method Id_ dry on page 23 Running 1 Remove the tips corresponding to well positions G12 and H12 from every tip box Method 1d_dry O COCR 00
25. 8 30 min 2 HOLD 90 20 min 3 25 cycles 94 15 sec 60 30 sec 51 396 ramp 30 sec 4 HOLD 99 10 min 5 HOLD 4 so At this point the OLA reaction is complete Proceed to Chapter 5 Purifying OLA Products Exonuclease SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 31 Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Method 1d dry Performing the OLA Reactions Biomek FX Method 1d dry About These Method 1d dry was developed for setups that have 1472 gDNA samples and 1 ligation Methods probe pool and uses use a single deck layout To minimize tip usage and prevent carryover contamination between quadrants these methods are designed so that the tips are positioned above the liquid level in the plates when dispensing reagents Preparing the 1 Thaw the SNPlex Oligonucleotide Ligation Kit components and probe pool at room Reagents temperature 2 Label a skirted 96 well PCR plate Assay Mix 3 Prepare the Assay Mix in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Automation Losses Totals Total Volume Reagent ee ee ee M Quadrant Nuclease Free Water 216 2 432 4 110 4 4002 0 SNPlex OLA Master Mix 235 0 470 0 120 0 4350 0 SNPlex Universal Linkers 48 plex 4 7 9 4 2 4 87 0 SNPlex dATP 4 7 9 4 2 4 87 0 SNPlex Probe Pool 500 nM 9 4 18 8 4 8 174 0 Total 470 0 940 0 240 0 8
26. B
27. ETHOD IS NON INFRINGING ALL OTHER WARRANTIES ARE EXPRESSLY DISCLAIMED YOUR USE OF THE METHOD IS SOLELY AT YOUR OWN RISK WITHOUT RECOURSE TO APPLIED BIOSYSTEMS TRADEMARKS Applied Biosystems ABI PRISM GeneMapper and MicroAmp are registered trademarks and AB Design Applera SNPlex and ZipChute are trademarks of Applera Corporation or its subsidiaries in the U S and or certain other countries GeneAmp is a registered trademark of Roche Molecular Systems Inc All other trademarks are the sole property of their respective owners Part Number 4360796 Rev B 6 2005 Contents Preface V How to Use This Guide iss oem ei j c cm D c Ron n V How to Obtain More Information 0 00 ee res Vi How to Obtain Support anaana naaa aaea eee et tees vi Safety and EMC Compliance Information vii Safety Conventions Used in This Document 0000 ee vii Chemical Waste Safety a anaana aaa Ix Introduction 1 GI ne yo ag Geary es Gee ee eae ee ae oe ee 2 Laboratory Design aana ce ee eee ee eee 4 Required Equipment and Materials aaa aaa es 5 Designing the Sample Plate Layout 0 0 0 cece ee 8 Setting Up the Biomek FX for Automating the SNPlex System Assay 9 OVEIVICW ous vxvad UP PPPESUSMUAETU re E YEN eee ees Sarees ed peg 10 Creating and Naming the Deck Layout 0 000 cece eee 12 micuilsohi 1BI e d rrnRm 13 Importing the Workspace File ll
28. EXv5 OLA PN S4 D bmt for Method la dry Or e Ib SNPLEXv5 OLA P4 SN D bmt for Method 1b dry 4 When the method pauses verify that the deck layout corresponds to the figure above and click OK to continue 5 After the method run is complete remove and discard the tip boxes and plates Mix1 to Mix4 6 Remove seal and briefly centrifuge the OLA reaction plates OLAI to OLA4 f Proceed to Thermal Cycling the OLA Reactions on page 27 26 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Methods 1a dry or 1b dry NET Thermal Cycling Thermal cycle the OLA plates using the following conditions the OLA Heactions Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 cycles 94 15 sec 60 30 sec 51 3 ramp 30 sec 4 HOLD 99 10 min 5 HOLD 4 Next Steps At this point the OLA reaction is complete Proceed to Chapter 5 Purifying OLA Products Exonuclease SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 2f Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Method 1c ary Performing the OLA Reactions Biomek FX Method 1c dry About the Method Method 1c dry was developed for setups that have 92 gDNA samples and 16 ligation probe pools The m
29. LA Protocol Wet gDNA Chapter 4 Performing OLA Using Wet gDNA c c Reaction Plate You can select from four Biomek FX methods depending on the number of samples and Layouts probe pools assayed in a single run The following table gives the plate layouts and the associated file for each layout The quadrant representation comes from the division of the 384 well reaction plates into four 96 well quadrants Although plate layouts are flexible some layouts are more efficient with reagent usage than others The layout that assays 1472 samples with a single probe pool is most efficient since reagent dead volume is limited to a single 96 well source container The layout that assays 92 samples with 16 probe pools is the most inefficient since reagent dead volume is spread across sixteen 96 well source containers For optimal use of SNPlex System reagent kits consider plate layouts batch sizes and gDNA setup dried vs wet Reference Performing the OLA Reactions Biomek FX Methods 1a wet or page 40 1b wet on Total Number of Samples 368 Number of Probe Pools Method File Name 1a SNPLEXv5 OLA PN S4 W bmt Plate 3 Plate 1 Control DNA NTC S 12239 98 ESESETES amp OOOOOOOO 8OOOOOOOOOOOOOOO00 amp OOOOOOOOOOOOOO00 SOOOOOOOOOOOOOO00 z0000000000000000 zo0o000000000000000 zoo0oo00000000000000 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 ZOOOOOOOOOOOOOOOO OOOOOOOOO
30. LA products E ES Exonuclease 5 a Elute ZipChute probes e Dilute purified OLA product iS S op gt oO Prepare sample plates 2 for electrophoresis Prepare PCR reactions Amplification Kit Create results groups and plate records Load and run sample plates Analyze data in GeneMapper software v3 5 1 64 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 7 Setting Up the PCR Reactions Preparing the Reagents Preparing the Reagents Preparing the 1 Thaw the SNPlex System Amplification Kit components at room temperature PCR Mix 2 Label a skirted 96 deep well plate PCR Mix 3 Label a 15 mL centrifuge tube 4 Combine the following volumes of reagents in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Losses Automation Totals Total Volume Total Volume Volume uL for Source Plate Reagent One 384 well Dead Volume Hansrer nary 4L for One uL for Four Platet uL t Excess uL 384 well 384 well H Plate Platestt Nuclease Free Water 909 9 287 2 198 1 1395 2 4125 0 SNPlex Amplification Master 1880 0 593 4 409 3 2882 7 8522 7 Mix SNPlex Amplification 188 0 59 3 40 9 288 3 852 3 Primers Total 2977 9 940 0 648 3 4566 2 13500 0 T 376 reactions per plate x volume for one reaction t 94tips in source plate x 10uL per tip Transfer losses for t
31. O OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO 0000000000000000 vOOOOOOOOOOOOOOOO time OOOOOOOOOOOOO SJ DOOOOOOOOOOOOO DOOOOOOOOOOOOO m QuwuOr 5xazzon amp OOOOOOOOQ o amp OOOOOOOOJO 9JO 9 N8OOOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 00O0OOOOOOOOOOOOO OOOOOOOOOOOOOOOO 00O0OOOOOOOOOOOOO 7OOOOOOOOOOOOOOOCO O0O0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 eO0000O0O0O0O0OOOOOOOO 00000000000O0OOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO sooo00000000000000 OOOOOOOOOOOOOOOO gm OOOOOOOOOOOOO x DOOOOOOOOOOOOO DOOOOOOOOOOOOO m Quwucor 5x a2zzonmn 77 Cc 56 e O0 O o o C o a O O O o x c S X C LL o 5 oW C t0 PB Do N O co v7 1c SNPLEXv5 OLA P16 1 D bmt Plate 3 Plate 1 C 5N e eH OOOOOO0 OOOOOOOOOOOOOOO OOOOOOOOOOOOOOO OOOOOOOOOOOOOO00 OOOOOOOOOCOOOOOO OOOOOOOOOOOOOOOO cOOOOOOOOOOOOOOO0 OO0OOOOOOOOOOOOO0 20000000000000000 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO eOOcOOOOOOOOOOOOOO OO0O0OOOOOOOOOOOOO OOOOOOOOOOOOOOO0 eOO0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO gi OOOOOOOOOOOOO eo6pooooooooooooo o00pooooooooooooo m QuwuOr 5x azzon 19 20 21 22 23 24 OOOOOO f EE HHHH OOOOO
32. O000 OOOOOOOOOOOOO000 cei c Co O 0000000000000000 0000000000000000 c i bN amp oO ci OOOOOOOOOOOOOOOO cOOOOOOOOOOOOOOOO ci Ea c Ka u OOOOOOOOOOOOO0000 OOOOOOOOOOOOO000 ce V e a oeOOOOOOOOOOOOOOOO oOOOOOOOOOOOOOOOO oO se Eos A O ES o 0000000000000000 20000000000000000 D on so x 2 uo Q g 0000000000000000 0000000000000000 bus Oo oO 3 un c OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO ae un un v 5 5 Q co OOOOOOOOOOOOOOOO ri j oooooooooooooooo a e 5 Oo O Oo 5 a D BOO OOOOOOOOOOOOO di IO OOOOOOOOOOOOO 3 D P ODM DA Z s SCAG 8g G 0 3 5 co 23 c eO r ap a a C3 doo S Qoo ci Q a O v o A oa go ans pe CO o ENr 6406 ce S 5 amp ON O8 Got SO E mo ngarag a 6 5 D 4509 Ho e E 2 o xw c QF a ge ogo oO s Se ES 2Be ad c 2o0otusx O 9 D oO S g ep O zo jem e RaQ D o O gt N A oa Sum amp Q un O m e e Un On o c gt ps o G 5u e amp O 4 amp E SD Ec O x e v o Qo gt e Q Q v 5 ue S S S e am A PE H DoZ e lt n ed Oo yo N Layouts probe pools assayed in a single run The following table gives the plate layouts and the the 384 well reaction plates into four 96 well quadrants 21 Reaction Plate You can select from four Biomek FX methods depending on the number of samples and amp 000ooooooQgjeoleoeeee amp 0000ooooopJjeoleogleeee amp Oooooooooljoejoeeeee amp O
33. OO OOOOOOOOOOOOOOO OOOOOOOOOOOOOOCO OOOOOOOOOOOOOO0O OOOOOOOOOOOOOOCO OOOOOOOOOOOOOO0O OOOOOOOOOOOOOOO OOOOOOOOOOOOOOCO OOOOOOOOOOOOOO0O OOOOOOOOOOOOOOO OOOOOOOOOOOOOO0O0 OOOOOOOOOOOOOOO 11 12 13 14 15 16 17 18 19 20 21 22 23 24 OOOOOOOOOOOO0 Oo O O O O O O O O O O O O O O O OOOOOOOOOOOOOOO00 eO000O0OOOOOOOOOOOO 0000000000000000 OOOOOOOOOOOOOOO0 0000O0OOOOOOOOOOO 0000O0O0O0O0O0O0OOOOOO OOOOOOOOOOOOOOOO time OOOOOOOOOOOOO EJ DOOOOOOOOOOOOO DOOOOOOOOOOOOO m QuwuOr 5xazzon Plate 4 Plate 2 Allelic Ladder lt Z O o c O O NTC amp OOOooooooe0 o0leeee 8OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO0 OO0OOOOOOOOOOOOOO TOOOOOOOOOOOOOOOO 000O0OOOOOOOOOOOO 0000O0OOOOOOOOOOO OOOOOOOOOOOOOOO00 O0OOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO0 eOO0OOOOOOOOOOOOOO 0000000000O0OOOOO OOOOOOOOOOOOOOOO0 00 00000000000000 soo0o00000000000000 vOOOOOOOOOOOOOOOO EO OOOOOOOOOOOOO o0poooooooooooooOo Oo06pooooooooooooo m QuuwuOor 5x azzoa amp OOOOO0O00 0 0 Oj6 amp OOOOOOOO JO 9jO 9je e e e S8OOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOOOO 00O0OOOOOOOOOOOOO 0O0OOOOOOOOOOOOOO0 TOOOOOOOOOOOOOOOO0 00O0OOOOOOOOOOOOO 0000000000O0OOOOO 0000000O0OOOOOOOO 0O0O0OOOOOOOOOOOOO0 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOO000
34. OOOO vOOOOOOOOOOOOOOOO0 ga OOOOOOOOOOOOO 60pooooooooooooo O06lpooooooooooooo m QuuOr 5xa2z2zon 00000000 00 00 0 0 amp Ooooooooocoejoojleeee 8OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOOOO SOOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 20000000000000000 z2o0o000000000000000 z zoo0oo00000000000000 z0000000000000000 zooooooo0000000000 zO000000000000000 OOOOOOOOOOOOOOO0 veOO0O0OOOOOOOOOOOOO 000000000O0O0OOOOO OOOOOOOOOOOOOOO0 eOO0OOOOOOOOOOOOOO s20000000000000000 YvOOOOOOOOOOOOOOOO gama OO OOOOOOOOO000 oo0pooooooooooooo O06poooooooooooooO m QuuiOr 5xazzon Method Number 1a wet 1b wet SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 38 t Z e O D O o D O O z eg D an St o 2 Q c lt O g Q gt ww D O 9 M aR o Y O t D O x G 2 o S s D w Mw aR Reference Performing the OLA Reactions Biomek FX Method 1c_wet on page 45 Total Number of Samples 92 Number of Probe Pools 16 Method File Name 1c SNPLEXv5 OLA P16 1 W bmt Plate 3 Plate 1 Control DNA Allelic Ladder NTC f 0000000 0 0 0 O18 OOO amp OoOooooooooe9joojleeee 8OOOOOOOOOOOOOOOO0 la0000000000000000 amp OOO
35. OOOOO OOOOOOOOOOOOOOOO0 YOOOOOOOOOOOOOOOO LA oO E Qo N eo E ga OOOOOOOOOOO0O00 time OOOOOOOOOOOOO N O 5oopooooooooooooo Of eo ooocooooooo0o00 za sr t O00pooooooooooooo t Oo06lpooooooooooooo a m QuwuiOr 5x azzon m QuuwuOor 5x azzoa 4 0 T A A O 55 OG Q a 1229992 SHE SEES amp OOOOOOOO 8OOOOOOOOOOOOOOO0 N8OOOOOOOOOOOOOOO00 amp OOOOOOOOOOOOOOOO amp 0000000000000000l amp OOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO 0000O0OOOOOOOOOOO 0O0O0OOOOOOOOOOOOO ce 0000O0OOOOOOOOOOO OOOOOOOOOOOOOOO0 TOOOOOOOOOOOOOOOO TOOOOOOOOOOOOOOO0 O 00O0OOOOOOOOOOOOO 000OOOOOOOOOOOOO E 000000O0O0O0OOOOOOO 000000000O0OOOOOO C OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 0000O0OOOOOOOOOOO 00O0OOOOOOOOOOOO0 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO O OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOO000 ob TT eOO0O0OOOOOOOOOOOOO 0000000000000000 V 00000000O0O0OOOOOO 00000000O0OOOOOOO E OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 0000O0OOOOOOOOOOO eOO0O0OOOOOOOOOOOOO aq D soo0oo00000000000000 soOo000000000000000 z 9000000000000000 a 0000000000000000 oO time OOOOOOOOOOOOO time O O00000000000 O f oobooooooooooooo 2 seopooooo0oo00000000 ab w I 0 po 000000000000 amp l oepooooooooooooo lt a QuwucoOr 5xazzoa m QuwuOr 5x azzoa LL A A eb X T OL o oO c SE v z Unique collection of 92 gDNA samples
36. OOOOOO00 0OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO 20000000000000000 0000000000000000 0000000000000000 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO s20000000000000000 OOOOOOOOOOOOOOO0 eo0pooooooooooooo O06pooooooooooooo m QuuOr 5xazzon Plate 4 H Allelic Ladder amp 800000000 J690 0 0 0 e e e amp OOoOoooo00JO 9jo 00 e e e amp 8OOOOOOOOOOOOOOO0O0 amp OOOOOOOOOOOOOOO0O amp OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOO0 00O0OOOOOOOOOOOOO0 000000O0O0OOOOOOOO OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 00O0OOOOOOOOOOOO00 eOOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOO00 OOOOOOOOOOOOOOO0 eOO0OOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 0000000000000000 12239 08 ESESETES amp OOOOOOOO OOOOOOOOOOOOOOO0 ga OOOOOOOOOOOOO0 8OOOOOOOOOOOOOOOO amp OOOOOOOOOOOOOO0O0O0 amp OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO 0OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 00000O0OOOOOOOOOO zOOOOOOOOOOOOOOOO 0O0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO OO0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO eOO0OOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOO0 ga OOOOOOOOOOOOO eopooooooooooooo oepooooooooooooo m QuuiOr 5x a2zzon Performing the OLA Reactions Biomek FX Methods 1a wet or page 40 1b wet on 3
37. OOOOOOOOOOO0O00 0000O0OOOOOOOOOOO OOOOOOOOOOOOOO0O00 TOOOOOOOOOOOOOO0O00 00000O0O0O0OOOOOOOO 0000O0OOOOOOOOOOO 20000000000O0OO0OOO O0OOOOOOOOOOOOO0O00 000O0OOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO eO0O0OOOOOOOOOOOO0O00 000000000O0OOOOOO OOOOOOOOOOOOOOOO0 eOOOOOOOOOOOOOOOO 00000000000O0O0OOO OO000000000000000 aD OOOOOOOOOOOOO nQuwuiOoOr 5x a zzon o0poooooooooooooOo O6lpooooooooooooo m amp OOOOOOOO OKILAK AA aoooooo0oo0oo000000000 amp OOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO 0O0OOOOOOOOOOOOO00 00000O0OOOOOOOOOO TOOOOOOOOOOOOOOO00 0000O0OOOOOOOOOOO 0000O0OOOOOOOOOOO 000000O0O0OOOOOOOO zoo0o00000000000000 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOO00 eO0O0OOOOOOOOOOOOO0O0 0000000000000000 o0oo0o00000000000000 eo000000000000000 sO000000000000000 0000000000000000 ga OOOOOOOOOOOOO o0pooooooooooooo O00pooooooooooooo m QuuwuOr 5x azzonu i ononooosseeteen o0 Plate 4 Plate 2 amp O0OoOoooooo0jleoooleeee amp Oooooooooe9joojleeee 8OOOOOOOOOOOOOOOO0 la0000000000000000 amp OOOOOOOOOOOOOO0O00 000OOOOOOOOOOOOO OOOOOOOOOOOOOO000 TOOOOOOOOOOOOOOO0 0000000O0O0OOOOOOO 00000O0OOOOOOOOOO 0000000000000O0OO 0O0OOOOOOOOOOOOOO0 000O0OOOOOOOOOOOO OOOOOOOOOOOOOOOO0 OOOOOOOOOOOOOOOO eO0O0OOOOOOOOOOOO0O00 00000000000O0O0O0OO OOOOOOOOOOOOOOOO eOO0OOOOOOOOOOOOOO 000000000000000
38. P4 Assay Mix P18 DNA5 to DNA8 P5 to P8 Uncovered tip P9 to P12 box P13 P14 Heinr LIll2lll eeoreoben 8 When the method pauses click OK to continue the method 9 When the method pauses remove the four used tip boxes in positions P9 to P12 and the DNA sample plates DNA5 to DNA8 from positions P5 to P8 10 Remove the tips corresponding to well positions G12 and H12 from four new uncovered tip boxes and place them and the plates on the deck as shown in the deck layout below Plate Position Method 1d_wet Deck Layout 3 OLA1 to OLAA P1 to PA Assay Mix P18 P13 DNAY to P5 to P8 DNA12 ay Uncovered tip P9 to P12 box i P15 NUNT Y rn 11 Click OK to continue the method SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 51 re Chapter 4 Performing OLA Using Wet gDNA 12 13 14 15 16 Performing the OLA Reactions Method 1d_wet When the method pauses remove the four used tip boxes in positions P9 to P12 and the DNA sample plates DNA9 to DNA12 from positions P5 to P8 Remove the tips corresponding to well positions G12 and H12 from four new uncovered tip boxes and place them and the plates on the deck as shown in the deck layout below Plate Position Method 1d_wet Deck Layout 4 OLA1 to OLAA P1 to P4 Assay Mix P18 SS E DNA13 to P5 to P8 MA DNA16 E Uncovered tip P9
39. SNPlex Genotyping System 48 plex AS Applied Automating OLA Using the Biomek FX M Biosystems GS Chapter 1 Getting Started Guide Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Copyright 2005 Applied Biosystems All rights reserved For Research Use Only Not for use in diagnostic procedures Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document This document is believed to be complete and accurate at the time of publication In no event shall Applied Biosystems be liable for incidental special multiple or consequential damages in connection with or arising from the use of this document NOTICE TO PURCHASER PLEASE REFER TO THE SNPlex GENOTYPING SYSTEM 48 PLEX USER GUIDE FOR LIMITED LICENSE OR DISCLAIMER INFORMATION Your installation and or use of the workspace and method files Method may affect the service coverage of your instrument under warranty or service contract Prior to installing and or using the Method check the warranty or service coverage of your instrument including limitations thereof or check with your service provider APPLIED BIOSYSTEMS MAKES NO WARRANTIES OF ANY KIND WHATSOEVER EXPRESS OR IMPLIED WITH RESPECT TO THE METHOD INCLUDING BUT NOT LIMITED TO WARRANTIES OF FITNESS FOR A PARTICULAR PURPOSE OR MERCHANTABILITY OR THAT THE M
40. User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPIex Genotyping System 48 plex General Automation Getting Started Guide 28 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Method 1c ary 5 Add 15 uL of the Assay Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells o ES S 000080000 000080000 Oe TIT TT I r Mix n h T 15 uL eoc000000 ec000000 000000080 00000000 00000000 NS Leave wells G12 and L N D o 3 lt 6 Label four 384 well plates containing dried gDNA OLAI to OLAA For information on the layout of dry gDNA plates see Method 1c dry on page 22 Running 1 Remove the tips corresponding to well positions G12 and H12 from every tip box Method 1c dry O OQ OQ Og O0 e e 000009 0000 pp 0000000000 9 6 6 0 0 0 0 0 Oo 000 00000009 600000 90000000000 OOOOOOOE OOOOOOG 90000 600000 Remove tips from wells G12 and H12 2 Place the uncovered tip boxes
41. annel pipettor 250 uL MLS Pipettors MLS Pipetting resevoirs 25 mL MLS Pipetting resevoirs 100 mL MLS Reaction ABI PRiSM 384 Well Clear Optical Applied Biosystems 4309849 Plates Reaction Plate with Barcode 50 plates ABI Prism 384 Well Optical Reaction Applied Biosystems 4326270 Plate with Barcode 500 plates SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 5 Chapter 1 Introduction Required Equipment and Materials Kits and Reagents Part Item continued Vendor N mber Reaction MicroAmp Full 96 Well Plate Cover Applied Biosystems N8010550 pale t ABI PRISM Optical Cover Compression Applied Biosystems 4312639 overs Pad e Heatseals Easy Peel individual sheets ABGene AB 0745 ane Easy Peel 610 meter roll ABGene AB 3739 sealers UNISEAL AL Whatman 7704 0002 Thermosealer ABGene AVB 0384 Plate Sealer ALPS 300 ABGene AB 0950 e Adhesive 384 Well Microplate Aluminum Sealing Corning 6569 seals Tape Adhesive PCR foil seal ABGene AB 0626 SILVERseal Greiner 676 090 Reagent reservoir Axygen RES SW96 HP Skirted 96 well PCR plates Axygen PCR 96 FS C Vortex MLS T IMPORTANT Applied Biosystems has found that certain plate covers negatively affect the performance of the SNPlex System assay If you use covers other than the recommended plate covers test them using the SNPlex System Control Set see
42. are of that robot For example the four methods provided for performing OLA using dry gDNA on the Biomek FX can be read only by the Biomek FX software Applied Biosystems provides a total of 11 methods for automating the pre PCR process Eight methods for the OLA setup four for using dry gDNA and four for using wet DNA One method for the exonuclease purification step One method for the OLA dilution step One method for the PCR setup step These methods are explained in succeeding chapters of this guide IMPORTANT The sample plate layouts and methods provided in this document are designed for experiments using batches of four 384 well plates Summary Setting up the Biomek FX for automating the OLA lab process involves five tasks Creating and Naming the Deck Layout on page 12 Framing the Deck on page 13 mporting the Workspace File on page 13 Copying the Method Files on page 15 e Reviewing the Supplied Methods on page 16 If you are using the Biomek FX instrument exclusively for the SNPlex System assay or if you are using the SNPlex System workspace for all other applications setting up the instrument is a one time process If you are using the instrument with other workspaces you may need to repeat some steps in the setup process such as importing the workspace file and reviewing the setup SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 11
43. art the method using 2 SNPLEXv5 EXO DILUTION bmt 4 When the method pauses click OK to continue 5 When the method next pauses remove and discard the eight used tip boxes SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 61 Running Biomek FX Method 3 Dilution Chapter 6 Diluting the Purified OLA Product 6 Remove tips corresponding to well positions G12 and H12 from eight new uncovered tip boxes and place them and the plates on the deck as shown in the deck layout below box Plate Position Deck Layout 2 OLA1 to OLA4 P5 to P8 Water P18 TL1 P1 Uncovered tip P9 to P16 OLA1 OLA P OLA OLA P4 Uu co J c2 n3 f Click OK to continue the method 8 After the method completes remove all eight used tip boxes and the water reservoir 9 Remove seal and briefly centrifuge the OLA reaction plates OLA1 to OLA4 Next Steps Store the plates if they will not be immediately used to prepare PCR reactions Otherwise return the plates to their positions on the deck to prepare PCR reactions Proceed to Chapter 7 Setting Up the PCR Reactions 62 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 7 setting Up the PCR Reactions J Introduction Setting Up the 4 Biomek FX for Automating the SNPlex System Assay
44. ate Layouts and Methods for the OLA Protocol Dry gDNA Performing the OLA Reactions Biomek FX Methods 1a_Dry or 1b_Dry Performing the OLA Reactions Biomek FX Method 1c_Dry or Performing the OLA Reactions Biomek FX Method 1d_Dry Chapter 3 Performing OLA Using Dry gDNA See page 20 See page 21 See page 24 See page 28 See page 32 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 19 Chapter 3 Performing OLA Using Dry gDNA Overview Overview About This This chapter provides information about automating the OLA protocol with the Biomek Chapter FX using disposable tips The procedures in this chapter assume the use of a batch of four SNPlex OLA reaction plates each containing 37 ng well of dried fragmented gDNA samples If you are using wet gDNA refer to Chapter 4 Performing OLA Using Wet gDNA Where You Are In the SN Plex Design and order System Assay SNPlex ligation probes Workflow s Design sample plate layout s Run PCR E 4 S fe fe E E l l e Prepare hybridization plates Oo Prepare gDNA a and bind PCR product to plates 2 Phosphorylate and Phosphorylate and E Add denaturant isolating 6 ligate probes ligate probes 2 o biolinylated strand on K linkers and linkers and i hybridization plate Oo gDNA OLA dry gDNA OLA wet oa gt Hybridize ZipChute M p
45. bout Workspace Instructions for automating the SNPlex System assay are contained in two types of files and Method Files workspace e method Workspace Files Workspace files contain labware and tip definitions pipetting templates techniques and liquid types They also contain settings needed by the Biomek FX software to run the method files The workspace file for the Biomek FX AB SNPLEX Workspace imp contains the following settings Setting Type Settings Labware Definitions snplex OLA ABI 384 in ACME Adapter snplex PCR ABI 384 in ACME Adapter snplex VWR 96 PP 16posALP snplex VWR 96 PP 1posALP snplex Axygen 96 DiaBot HP Axygen96 50uL Barrier Pipetting Templates Default Template Low Volume Techniques snplex AssayMix 3uL snplex AssayMix 5uL snplex DNA 2uL snplex ExoMix 5uL snplex ExoDil 5uL snplex PCRMix 7 92uL snplex OLARxn 2 08uL Liquid Types snplex DNASample snplex ExoMix snplex AssayMix snplex OLARxn snplex PCRMix Tips AxyP50 Barrier 10 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Overview Method Files Method files contain the instructions for a workflow activity on a robot Method files are specific for a given robot and can be read only by the softw
46. btain Support To contact Applied Biosystems Technical Support from North America by telephone call 1 800 899 5858 For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities vi SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Safety and EMC Compliance Information Safety Conventions Used in This Document Safety Alert Four safety alert words appear in Applied Biosystems user documentation at points in Words the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below Definitions IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical N eue Indicates a potentially hazardous situation that if not avoided may result in minor
47. c wet 1d wet this Guide are _dry in f a 96 well DNA source plate are reserved for 1 control DNA 1 ions o Dry gDNA f 92 gDNA samples 4 pos ing ion o llect ique co S Un NTC and 2 allelic ladder wells Total number of samples in a batch containing four 384 T Method numbering reflects methods provided for other SNPlex System assay protocols Methods 1a dry through 1d described in Chapter 3 Performing OLA Us P Probe Pool Does not include control DNA or NTC well plates 39 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Methods 1a wet or 1b wet Performing the OLA Reactions Biomek FX Methods 1a wet or 1b wet About These Methods Preparing the Reagents 40 Methods la wet and 1b wet were developed for setups that have 368 gDNA samples and four probe pools These methods use identical three deck layouts The difference between them is the pattern in which the instrument dispenses the reagents and DNA samples into the wells of the OLA reaction plates In Method la wet e Assay Mix from source plate Mix is transferred to all four quadrants of OLA reaction plate OLA1 Mix 2 to OLA2 Mix 3 to OLA3 and Mix 4 to OLA4 e Samples from source plate DNA1 are transferred to quadrant 1 of all four OLA plates OLA1 to OLA4 DNA2 to quadran
48. e RR ROREM 65 Running Biomek FX Method 4 PCR 2 0 0 eee In 66 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Preface How to Use This Guide Purpose of This This guide provides a representative workflow using the SNPlex Genotyping System Guide 48 plex with the Biomek FX system It provides information to assist you in automating the Oligonucleotide ligation assay OLA using the Biomek FX instrument and is intended to be used with the SNPlex Genotyping System 48 plex User Guide PN 4360856 Audience This guide is written for principal investigators and laboratory staff who intend to use the SNPlex Genotyping System 48 plex with robotics Assumptions This guide assumes that you have read the SNPlex Genotyping System 48 plex User Guide and the SNPlex Genotyping System 48 plex General Automation Getting Started Guide PN 4363143 and that you have a working knowledge of the assays and methods used for the SNPlex Genotyping System 48 plex Text Conventions Bold indicates user action For example Type 0 then press Enter for each of the remaining fields e Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix e A right arrow bracket gt separates successive commands you select from a drop down or shortcut menu For example Select File gt Open gt Spot Set User Attention Two user attention words a
49. e imp then click Import 14 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Copying the Method Files Copying the Method Files 1 Start the Biomek FX software 2 Select File gt Open browse to the location to which you unzipped the method files and select a method file 3 Select File gt Save to save the file to an appropriate location 4 Repeat steps 3 to 5 until you have saved the following files 1a SNPLEXv5 OLA PN S4 D bmt 1c SNPLEXv5 OLA P16 1 W bmt 1b SNPLEXv5 OLA P4 SN D bmt 1d SNPLEXv5 OLA P1 S816 W bmt 1c SNPLEXv5 OLA P16 S1 D bmt 2 SNPLEXv5 EXO bmt 1d SNPLEXv5 OLA P1 16 D bmt 3 SNPLEXv5 EXO DILUTION bmt 1a SNPLEXv5 OLA PN S4 W bmt 4 SNPLEXv5 PCR bmt 1b SNPLEXv5 OLA P4 SN W bmt Note You can also use Windows Explorer to copy the files from the location to which you downloaded them to an appropriate location on the Biomek FX workstation SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 15 Reviewing the Supplied Methods Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Reviewing the Supplied Methods Verifying the Because the proper operation of a method depends on the presence of specific conditions Method such as the number of 384 well reaction plate
50. e of the recommended plate covers listed in Table 1 3 on page 1 9 of the SNPlex Genotyping System 46 plex User Guide If you use covers other than the recommended plate covers test them using the SNPlex System Control Set see Appendix A of the SNPlex Genotyping System 48 plex User Guide 4 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 1 Introduction Required Equipment and Materials Required Equipment and Materials Equipment Part Item Vendor Nnnber GeneAmp PCR System 9700 Dual 384 Well Sample Contact your Applied Biosystems Block Module representative for information Biomek FX Single Arm System with Contact your Beckman Coulter Hepresentative e 96 channel 20 uL disposable tip pipetting head 719367 e Disposable tip loader ALP 719356 Beckman Coulter e 2 Standard single position ALPs 719357 e 16 position 4X4 high density ALP 719360 Centrifuge equipped to accommodate reaction plates Major Laboratory Supplier MLS Sealer for microtiter plates Recommend e ALPS 300 Heat Sealer AB 0950 e Air compressor with clean air package ABGene CMP 950 e Easy Peel Foil Sealing Film for ALPS 300 AB 3739 Consumables and Small Equipment Part Item Vendor Number 15 mL centrifuge tubes MLS 384 well plate adapters eight Acme Automation CR7019 50 uL Barrier 96 channel disposable tip boxes Axygen FXF 50 L R S Multich
51. ethod transfers Assay Mix from 16 plates into four OLA reaction plates in four steps each corresponding to a deck layout e Assay Mix from source plates Mix to Mix4 is transferred to plate OLAT Assay Mix from source plates Mix5 to Mix8 is transferred to plate OLA2 Assay Mix from source plates Mix9 to Mix12 is transferred to plate OLA3 Assay Mix from source plates Mix13 to Mix16 is transferred to plate OLAA Preparing the 1 Thaw the SNPlex Oligonucleotide Ligation Kit components and probe pools at Reagents room temperature 2 Label 16 skirted 96 well PCR plates Mix1 to Mix16 3 Label 16 15 mL centrifuge tubes Mix to Mix16 4 For each of the 16 SNPlex Ligation Probe Pools prepare an Assay Mix in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Automation Losses Totals Reagent Volume uL for Source Plate Transfer Loss Coo g One Quadrant Dead Volumet Excess uL H 5 Quadrant Nuclease Free Water 216 2 432 4 110 4 759 0 SNPlex OLA Master Mix 235 0 470 0 120 0 825 0 SNPlex Universal Linkers 48 plex 4 7 9 4 2 4 16 5 SNPlex dATP 4 7 9 4 2 4 16 5 SNPlex Probe Pool 500 nM 9 4 18 8 4 8 33 0 Total 470 0 940 0 240 0 1650 0 94 tips in source plate X 10uL per tip un H Transfer losses for transferring bulk mixture into 96 well source plate Use the volumes in this column to prepare the reagents Refer to the SNPIex Genotyping System 48 plex
52. eviewing the Supplied Methods Verifying the Methods also require the use of specific labware in order to function properly The Labware following table lists the labware used to develop the Biomek FX methods Description Vendor Title in Method 384 well reaction plate Applied Biosystems OLA_ABI_384_in_ACME_Adapter SOMME PCR ABI 384 in ACME Adapter Axygen 50 uL barrier tips Axygen Axygen96 50uL Barrier FXF 50 L R S Reagent reservoir Axygen Axygen 96 DiaBot HP RES SW96 HP Skirted 96 well PCR plate Axygen VWR_96_PP_16posALP iix 2 VWR 96 PP 1posALP Once the setup is complete you can start using the Biomek FX instrument for automating the OLA lab protocols of the SNPlex System assay as described in the following chapters SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 17 Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Reviewing the Supplied Methods 18 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Introduction Setting Up the Biomek FX for Automating the SNPlex System c 3 Performing OLA e Performing OLA a Using Dry DNA S Using Wet DNA Purifying OLA 22 225 Products Exonuclease c Diluting the Purified comes 7 OLA Product Setting Up the PCR Reactions Notes Overview Pl
53. fuge the plate briefly to collect the contents at the bottom of the wells Mix 1 Y l00000000000 00000000000 30 uL 00000000000 000000000000 0eo0000000000 eo0000000000 900000000000 i00000000000 Leave wells G12 and H12 empty 6 Label four 384 well plates containing dried down gDNA samples OLA1 to OLA4 For information on the layout of dry gDNA plates see Method la dry on page 21 or lb dry on page 22 SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 25 Chapter 3 Performing OLA Using Dry gDNA Performing the OLA Reactions Biomek FX Methods 1a dry or 1b ary Running 1 Remove the tips corresponding to well positions G12 and H12 from every tip box Methods 1a dry and 1b dry 000p 6600 pp 6600000000 6 0600 000 0 0Q 0000000000009 0 00 00000 0Q 000000000000 00000000 000 0 000 0 0 00000000 ooo o000t gt ee 000009 Remove tips from wells G12 and H12 2 Place the uncovered tip boxes and plates on the deck as shown in the following figure placing an ACME adapter underneath each OLA plate Plate Position Methods 1a_dry and 1b_dry Deck Layout 1 OLA1 to OLA4 P1 to P4 Mix1 to Mix4 P5 to P8 Uncovered tip P9 to P12 box OLA ES Pl om C 3 Start the method using either e la SNPL
54. ix4 24 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Performing the OLA Reactions Biomek FX Methods 1a dry or 1b_dry Chapter 3 Performing OLA Using Dry gDNA 4 For each of the four SNPlex Ligation Probe Pools prepare an Assay Mix in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Losses AT elu Cr Totals Volume uL for Source Plate Transfer Loss Total Volume uL neagent One Quadrant Dead Volumet Excess uL uel al Quadrants Nuclease Free Water 216 2 432 4 110 4 1407 6 SNPlex OLA Master Mix 235 0 470 0 120 0 1530 0 SNPlex Universal Linkers 48 plex 4 7 9 4 2 4 30 6 SNPlex dATP 4 7 9 4 2 4 30 6 SNPlex Probe Pool 500 nM 9 4 18 8 4 8 61 2 Total 470 0 940 0 240 0 3060 0 94 tips in source plate x OuL per tip Qo Transfer losses for transferring bulk mixture into 96 well source plate Use the volumes in this column to prepare the reagents Refer to the SNPIex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide 5 Add 30 uL of the Assay Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centri
55. ization plate Oligonucleotide Ligation Kit o a Z xe G a o Ww ez ab D v 9 oc G 9 w N T um gt T Hybridize ZipChute probes Purify OLA products Exonuclease Chapter 5 Purification Kit Elute ZipChute probes Dilute purified OLA product Chapter 6 Assay Standards Kit Dispense allelic ladders Prepare PCR reactions Chapter 7 Amplification Kit Create results groups and plate records Load and run sample plates Analyze data in GeneMapper software v3 7 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 3 Chapter 1 Introduction Laboratory Design Laboratory Design The protocols contained in this guide should be performed in an amplicon free OLA lab such as that shown in the following figure OLA Lab Layout SY SS Plate Centrifuge Sealer This is a vertical shelf unit depending on the number of shelves can house 2 thermal cyclers per shelf Optional For additional information about laboratory design refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide Plate Sealing A plate sealer is recommended but not required Applied Biosystems has found that certain plate covers negatively affect the performance of the SNPlex System assay If you do not use a plate sealer you may use on
56. l and store the OLA reaction plates 10 Remove seal and briefly centrifuge the PCR plates PCRI to PCR4 Next Steps At this point move the PCR plates to the PCR lab to complete thermal cycling and the rest of the steps in the SNPlex System assay workflow IMPORTANT Never move equipment containers or other items from the PCR Laboratory or data collection area into the OLA Laboratory OLA PCR Lab Lab OLA 4 Z7 PCR Lab A Lab SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 67 Chapter 7 Setting Up the PCR Reactions Running Biomek FX Method 4 PCR 68 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Applera is committed to providing the world s leading technology and information for life scientists Applera Corporation consists of the Applied Biosystems and Celera Genomics businesses Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 06 2005 Applied Biosystems Part Number 4360796 Rev
57. lelel I 13 Copying the Method Files ellllellllel eens 15 Reviewing the Supplied Methods llle 16 Performing OLA Using Dry gDNA 19 eU AM 20 Plate Layouts and Methods for the OLA Protocol Dry gDNA 21 Performing the OLA Reactions Biomek FX Methods 1a dry or 1b dry 24 Performing the OLA Reactions Biomek FX Method 1c dry 28 Performing the OLA Reactions Biomek FX Method 1d dry 32 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX iii Performing OLA Using Wet gDNA 35 OVOlIVIB Wu x 3 edad os ae eh D eR p e CIR adie ad RR oe Sea CAD Red poto 36 Plate Layouts and Methods for the OLA Protocol Wet gDNA 37 Performing the OLA Reactions Biomek FX Methods 1a wet or 1b wet 40 Performing the OLA Reactions Biomek FX Method 1c wet 45 Performing the OLA Reactions Method 1d wet 49 Purifying OLA Products Exonuclease 53 OVERVIEW a s sinh cit b E wed a ue ene a a ae Nake a oap ta a apo E 54 Running Biomek FX Method 2 Exo nananana anaana eee ete 55 Diluting the Purified OLA Product 59 OVEN a eae apne aie ano ee aha ease eae dm du ns Ee Ee ee Ege ORE 60 Running Biomek FX Method 3 Dilution 0 0 ee 61 Setting Up the PCR Reactions 63 GaU PED Phin ce ai te oe tk tes ea aa PR ee ee a Ne oe es 64 Preparing the Reagents s dose wx ad RERO week eee Ee ee
58. nd health regulations Safety and EMC Compliance Information Chemical Waste Safety X SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 1 Introduction Introduction See page 2 Overview Setting Up the Biomek FX for Automating the SNPlex System Assay Laboratory Design Let ea c Performing OLA c 2 9 Performing OLA Required Equipment See page 5 NC Using Dry DNA 7 NZ Using Wet DNA g Dry g and Materials Designing the See page 8 Sample Plate Layout I Purifying OLA gow 422 z Products Exonuclease gt ___Diluting the Purified e OLA Product mp Setting Up the PCR Reactions Notes SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX 1 Chapter 1 Introduction Overview Overview The SNPlex System assay consists of several protocols which involve manipulating small volumes between 96 and 384 well plates and reservoirs This guide contains the protocols required to complete the oligonucleotide ligation assay OLA portion of SNPlex System assays using the Biomek FX System About This Guide The SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide provides instructions for automating the OLA protocols using The Biomek FX System For information about automating the OLA procedures usi
59. ng on the number of samples and probe pools assayed in a single run Arraying gDNA in 96 Well Plates Array your gDNA samples in skirted 96 well PCR plates 92 samples per plate Reserve well E12 for the SNPlex System kit control gDNA and well F12 for the No Template Control NTC Leave wells G12 and H12 empty these wells are used as Allelic Ladder wells during analysis For example you may array 368 gDNA samples among four 96 well plates as shown DNA Source Plate 1 DNA Source Plate 3 BOOT GIO DIUINO a9 3 Gm G9 Gr amp e9 Gas Ge Gad Q8 e Grd amp 2 10 t8 86 80 9 Go 88 8 n5 89 89 B ee e Qo G9 C19 a8 6 G42 Cm 58 68 79 e 8 ro Q9 87 85 amp 6 89 6 9 8 8m c er 9 Gos Gr Gs G2 Gas e ED Qs Ce Grd o 4 2 5 a0 8 9 2 80 ED r9 04 82 D es 98 Qo Ed Ged e GD Gad E 6e Qe Ge 8 9 2 9 8 9 89 8 8 r7 9 9 E eg e Go 13 Ge Ge Gaz Gas Ed e amp amp 9 v FO Q9 2 80 8 e 84 amp r9 r9 66 V F a0 a9 Go G14 G2 Gan Gd Qa 69 e G9 V amp 0 G9 e 8 69 8 amp 5 69 60 a e 499 Go 18 Ge e Ed Ger 69 66 Gri O 9 9 5 8 r2 6 6 GS H 492 00 Go G9 28 a2 a0 Gud Gd G9 G2 89 e o9 5 BH WM HH ey 79 OH Gr 689 Qe Gat Gos 61 Ge 659 Gat 9 Ger G8 B
60. ng other instruments refer to the appropriate guide See Documentation on page 7 The 384 well protocol For information about the 96 well protocol refer to the SNPlex Genotyping System 48 plex User Guide PN 4360856 for instructions The Applied Biosystems 3730x DNA Analyzer to collect data GeneMapper software v3 7 to analyze data Assumptions This guide assumes that you have read the SNPlex Genotyping System 48 plex User Guide and the SNPlex Genotyping System 48 plex General Automation Getting Started Guide PN 4363143 and that you have a working knowledge of the assays and methods used for the SNPlex Genotyping System 48 plex This guide also assumes that you have a working knowledge of the operation of the Biomek FX including set up framing and building of the deck 2 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 1 Introduction Overview SNPlex System The following diagram illustrates the SNPlex System workflow Automation Workflow Design sample plate layout Prepare gDNA iced Run PCR Set up liquid handling instrument Chapter 2 Prepare hybridization plates PCR Laboratory and bind PCR product to plates OLA Laboratory Phosphorylate and Phosphorylate and ligate probes linkers ligate probes linkers and gDNA OLA dry and gDNA OLA wet Chapter 3 Chapter 4 Add denaturant isolating biotinylated strand on hybrid
61. od 1c wet was developed for setups that have 92 gDNA samples and 16 ligation probe pools The method transfers DNA samples and 16 Assay Mixes into four OLA reaction plates in four steps each corresponding to a deck layout DNA samples are transferred to all OLA plates then Assay Mix from source plates Mix to Mix4 is transferred to plate OLAT e Assay Mix from source plates Mix5 to Mix is transferred to plate OLA2 Assay Mix from source plates Mix9 to Mix12 1s transferred to plate OLA3 Assay Mix from source plates Mix13 to Mix16 is transferred to plate OLAA Preparing the 1 Label 16 skirted 96 well PCR plates Mix1 to Mix16 Reagents 2 Label 16 15 mL centrifuge tubes Mix to Mix16 3 Label a DNA source plate DNA Sample For information on setting up DNA source plates see Arraying gDNA in 96 Well Plates on page 37 4 For each of the 16 SNPlex Ligation Probe Pools prepare an Assay Mix in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Losses Automaton Totals Total Volume Reagent eae ees mn E i Quadrant Nuclease Free Water 28 2 94 0 21 8 144 0 SNPlex OLA Master Mix 235 0 783 3 181 7 1200 0 SNPlex Universal Linkers 48 plex 4 7 15 7 3 6 24 0 SNPlex dATP 4 7 15 7 3 6 24 0 SNPlex Probe Pool 500 nM 9 4 31 3 7 3 48 0 Total 282 0 940 0 218 0 1440 0 94 tips in source plate x 10uL per tip On H Transfer losses for transferring
62. ooooooo0Qnloejoejleeee qNOOOOOOOOOOOOOOOO qgOOOOOOOOOOOOOOOO amp Ooooooooooooooool NOOOOOOOOOOOOOOO0 amp SOOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO 9 0OOOOOOOOOOOOOOO 0OOOOOOOOOOOOOOOO ce CEOOOOOOOOOOOOOOOO CEOOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO O 0000000000000000 0O0O0OOOOOOOOOOOOOO P O00OOOoOOoOoOoOoooooooOo OOOOOOOOOOOOOOOO Cy 2000000000000000 2000OOOOOOOOOOOOO OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO zOOOOOOOOOOOOOOOO zOOOOOOOOOOOOOOOO Sr 2OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO e ob oOOOOOOOOOOOOOOOO 0000000000000000 V 0000000000000000 0000000000000000 2 OOOOOOOOOOOOOOOO OOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO eOOOOOOOOOOOOOOOO o c Z 0000000000000000 o6000O0OOOOOOOOOOOO un z 70000000000000000 qQ 0000000000000000 al o imm OOOOOOOOOOOOcO o iO OOOOOOOOOOO00 lt wy o t E Z iL n a lt b x oL o o c E a 5 Plate Layouts and Methods for the OLA Protocol Dry gDNA SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Plate Layouts and Methods for the OLA Protocol Dry gDNA C Z e O e O O E o D O O E z ge D an ise X ie Q lt O Go c Reference Performing the OLA Reactions Biomek FX Methods 1a dr y or 1b dry on page 24 Total Number of Samples 368 Number of Probe Pools Method File Name 1b SNPLEXv5 OLA P4
63. or moderate injury It may also be used to alert against unsafe practices ANITY Indicates a potentially hazardous situation that if not avoided could result in death or serious injury Chemical Hazard Nene CHEMICAL HAZARD Before handling any chemicals refer to Warning the Material Safety Data Sheet MSDS provided by the manufacturer and observe all relevant precautions About MSDSs Chemical manufacturers supply current Material Safety Data Sheets MSDSs with shipments of hazardous chemicals to new customers They also provide MSDSs with the first shipment of a hazardous chemical to a customer after an MSDS has been updated MSDSs provide the safety information you need to store handle transport and dispose of the chemicals safely Each time you receive a new MSDS packaged with a hazardous chemical be sure to replace the appropriate MSDS in your files SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide vii Safety and EMC Compliance Information Safety Conventions Used in This Document Obtaining You can obtain from Applied Biosystems the MSDS for any chemical supplied by MSDSs Applied Biosystems This service is free and available 24 hours a day To obtain MSDSs 1 2 Go to https docs appliedbiosystems com msdssearch html In the Search field type in the chemical name part number or other information that appears in the MSDS of interest Select the language of yo
64. otals Total Volume Volume uL for Source Plate Transfer Loss neageni One Quadrant Dead Volumet Excess uL Eor te Quadrants Nuclease Free Water 28 2 94 0 21 8 567 0 SNPlex OLA Master Mix 235 0 783 3 181 7 4725 0 SNPlex Universal Linkers 48 plex 4 7 15 7 3 6 94 5 SNPlex dATP 4 7 15 7 3 6 94 5 SNPlex Probe Pool 500 nM 9 4 31 3 7 3 189 0 Total 282 0 940 0 218 0 5670 0 94 tips in source plate x 10uL per tip Cr H Transfer pipetting losses for transferring bulk mixture into 96 well source plate Use the volumes in this column to prepare the reagents Refer to the SNPlex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 49 4 5 Running 1 Method 1d wet 2 3 Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Method 1d wet Add 58 uL of the Assay Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells Assay Mix j 58 uL 0000000 000000080 000000060 00000000
65. per software v3 7 54 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Running Biomek FX Method 2 Exo Assumptions About this Method Preparing the Exonuclease Mix Chapter 5 Purifying OLA Products Exonuclease Running Biomek FX Method 2 Exo comer G This method assumes that four 384 well OLA reaction plates prepared in Chapter 3 or Chapter 4 have been prepared and thermalcycled This method uses two deck layouts The first deck layout adds Exonuclease mix to the OLA reaction plates OLA1 and OLA2 The second deck layout adds Exonuclease mix to OLA reaction plates OLA3 and OLA4 The well contents are mixed after each dispense of the Exonuclease mix 1 Prepare an Exonuclease mix in a 15 ml centrifuge tube and mix thoroughly Automated Manual Automation Losses Automation Totalstt Total Volume Total Volume Volume uL for Source Plate f Transfer Loss uL for One uL for Four Heagent My era Br Excess uL 5 384 well 384 well H Plate Plates Nuclease Free Water 1579 2 789 6 453 6 2822 4 7560 0 SNPlex Exonuclease Buffer 188 0 94 0 54 0 336 0 900 0 SNPlex Lamda Exonuclease 75 2 37 6 21 6 134 4 360 0 SNPlex Exonuclease 37 6 18 8 10 8 67 2 180 0 Total 1880 0 940 0 540 0 3960 0 9000 0 T 376 reactions per plate x volume for one reaction t 94tips in source plate x 10uL per tip Transfer losses for transfer
66. ppear in Applied Biosystems user documentation Each word Words implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide V Preface How to Obtain More Information Examples of the user attention words appear below Note The size of the column affects the run time IMPORTANT To verify your client connection to the database you need a valid Oracle user ID and password How to Obtain More Information For more information about using the SNPlex Genotyping System 48 plex refer to the SNPlex Genotyping System 48 plex User Guide PN 4360856 SNPlex Genotyping System 48 plex Quick Reference Card PN 4360855 SNPlex Genotyping System 48 plex Assay Design and Ordering Guide PN 4357460 SNPlex Genotyping System 48 plex General Automation Getting Started Guide PN 4363143 SNPlex Genotyping System 48 plex Automating PCR Using the Tomtec Quadra 3 Getting Started Guide PN 4358100 Send Us Your Applied Biosystems welcomes your comments and suggestions for improving its user Comments documents You can e mail your comments to techpubs appliedbiosystems com How to O
67. ransferring bulk mixture into 96 well source plate tt Use the volumes in this column to prepare the reagents Refer to the SNPlex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide 5 Add 140 uL of the PCR master Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells Mix 1 L 140 uL 000000080 000000080 000000080 00000000 00000000 OOOO OOO a I Leave wells G12 and H12 empty 6 Label four 384 well ABI PRISM clear optical reaction plates PCR1 to PCR4 SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 65 Chapter 7 Setting Up the PCR Reactions Running Biomek FX Method 4 PCR Running Biomek FX Method 4 PCR About this This method uses two deck layouts The first deck layout adds PCR mix to all PCR Method reaction plates PCR1 to PCR4 and transfers aliquots from OLA1 and OLA2 to PCRI and PCR 2 respectively The second deck layout transfers aliquots from OLA3 and OLA4 to PCR3 and PCRA respectively Running the 1 Remove the tips corresponding to well positions G12 and
68. ring bulk mixture into 96 well source plate tt Use the volumes in this column to prepare the reagents Refer to the SNPlex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPlex Genotyping System 48 plex General Automation Getting Started Guide 2 Add 90 uL of the Exonuclease mix into each well of a skirted 96 well plate excluding G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells Mix 1 90 uL 00000000 00000000 00000000 000000080 OCOOOCOOO Leave wells G12 an 2 an _ N D E o0 m x SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 55 Chapter 5 Purifying OLA Products Exonuclease Running Biomek FX Method 2 Exo Running This 1 Remove the tips corresponding to well positions G12 and H12 from every tip box Method Remove tips from wells G12 and H12 2 Place eight uncovered tip boxes and plates on the deck as shown in the following figure placing an ACME adapter for the Thermal Exchange ALP underneath each OLA plate Plate Position Deck Layout 1 OLA1 to OLAA P5 to P8 Exo Mix P18 Uncovered tip P9 to P16 box 9 E 3 Start the method using 2 SNPLEXv5 EXO bmt
69. robes o Purify OLA products E Exonuclease E l E fat Elute ZipChute probes e Dilute purified OLA product S S Prepare sample plates 2 S for electrophoresis w Prepare PCR reactions i a E lt x Create results groups and plate records Load and run sample plates Analyze data in GeneMapper software v3 7 20 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide z Number of Probe Pools 4 CH O O T co Q p E ac c 2 _ N S BT tz o9 ke 2 nV Vw cO lt 5 gt eogt Zs D Z O ran 5 c d OD ug CC e oO t S otu amp us 8 ps g m zi 2 o S B Sg e X954 Zo 3 a SO2ES 9 Ooo5 9 z a gt D cm 6 00 aS cb O c O z 0s 43 O gt d o 2 0 oc gt D D O ci Un O c cx c Q f D O c 0 a u O 2 E O o a eo 0 c 800000000 300000000 g So DS XR a aea 800000000 O Q O H un u p gt 8OOOOOOOOOOOOO0O000 8OOOOOOOOOOOO0O0000 D M oO am e Hes oO O eb p amp OOoooooooooooooool NOOOOOOOOOOOOOOO0 A C z _ e wu KO SOOOOOOOOOOOOOOO0 amp OOOOOOOOOOOOOOOO lt Q OD of I N ci JN D EO OO CORO SOOO eo Eo OOOO CU COU DOO B ca coo n 2 0 0000000000000000 0000000000000000 O e ON cei e to T OOOOOOOOOOOOOO000 OOOOOOOOOOOOOO000 O L 5 o a 70000000000000000 70000000000000000 O _ e O OOOOOOOOOOOOO
70. rom wells G12 and H12 Place the five uncovered tip boxes and plates on the deck as shown in the deck layout below Plate Position Method 1c_wet Deck Layout 1 OLA1 to OLAA P1 to PA DNA sample P18 ix Mix1 to Mix4 P5 to P8 Uncovered tip P9to P12 box P17 i E L E N Samples ES L Start the method using lc SNPLEXv5 OLA P16 S1 W bmt When the method pauses remove and discard the four used tip boxes from P9 to P12 and plates Mix to Mix4 from P5 to P8 46 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Method 1c_wet 5 Remove the tips corresponding to well positions G12 and H12 from four new tip boxes and place them and the plates as shown in the deck layout below Plate Position Method 1c wet Deck Layout 2 OLA1 to OLAA P1 to P4 DNA sample P18 SS Mix5 to Mix8 P5 to P8 Uncovered tip P9 to P12 P14 box eters tee bebe EIE HE pis DN Samples d 6 Click OK to continue the method f When the method pauses remove the four used tip boxes in positions P9 to P12 and the Assay Mix plates Mix5 to Mix8 from positions P5 to P8 8 Remove the tips corresponding to well positions G12 and H12 from four new tip boxes and place them and the plates as shown in the deck layout below Pla
71. s you should verify that the methods you Conditions copied have the specifications listed in the following table Number of MUSETO Mene Method File Name 384 well Besdeni Toa he Tip DUIOSE d Source Reservoirs mm ss Boxee Plates 1a dry 1a SNPLEXv5 OLA PN S4 D bmt 4 4 4 16 4 1b dry 1b SNPLEXv5 OLA P4 SN D bmt 4 4 4 19 4 1c dry 1c SNPLEXv5 OLA P16 S1 D bmt 4 16 11 25 16 1d dry 1d SNPLEXv5 OLA P1 S16 D bmt 4 1 2 24 1 1a wet 1a SNPLEXv5 OLA PN S4 W bmt 4 8 15 31 20 1b_wet 1b SNPLEXv5 OLA P4 SN W bmt 4 8 15 32 20 1c wet 1c SNPLEXv5 OLA P16 S1 W bmt 4 17 13 48 17 1d wet 1d SNPLEXv5 OLA P1 S16 W bmt 4 17 13 49 17 2 2 SNPLEXv5 EXO bmt 4T 1 12 39 16 3 3 SNPLEXv5 EXO DILUTION bmt 41 1 13 08 16 4 4 SNPLEXv5 PCHR bmt 41 4 1 15 22 17 T Reaction plates are the output plates that are carried over from the previous method 16 Note Four methods are used to complete the OLA process Select one of the eight OLA setup methods that is la dry la wet 1b dry lb wet and so forth The four OLA reaction plates are carried over into method 2 Exo then into method 3 Dilution and then into method 4 PCR In method 4 aliquots from the original four reaction plates are transferred into four new PCR reaction plates SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay R
72. t 2 DNA3 to quadrant 3 and DNA4 to quadrant 4 In Method 1b wet e Assay Mix from source plate Mix 1 is transferred to quadrant 1 of all four OLA reaction plates OLA1 to OLA4 Mix 2 to quadrant 2 Mix 3 to quadrant 3 and Mix 4 to quadrant 4 e Samples from source plate DNA1 are transferred to all wells of plate OLA1 DNA2 to OLA2 DNA3 to OLA3 and DNA4 to OLAA 1 Thaw the SNPlex Oligonucleotide Ligation Kit components and probe pools at room temperature 2 Label four skirted 96 well PCR plates Mix to Mix4 3 Label four 15 mL centrifuge tubes Mix1 to Mix4 4 Label four empty 96 well plates for use as DNA source plates DNAI to DNA4 For more information on DNA source plate layouts see Plate Layouts and Methods for the OLA Protocol Wet gDNA on page 37 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Performing the OLA Reactions Biomek FX Methods 1a wet or 1b_wet Chapter 4 Performing OLA Using Wet gDNA 5 For each of the four SNPlex Ligation Probe Pools prepare an Assay Mix in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Losses AT elu Cr Totals Volume uL for Source Plate Transfer Loss Total Volume uL Reagent t i for Four One Quadrant Dead Volume Excess uL 8 Quadrants Nuclease Free Water 28 2 94 0 21 8 228 6 SNPlex OLA Master Mix 235 0 783 3 181 7 1905 0 SNPlex Universal Linkers 48 ple
73. t Utility SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 13 Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay Importing the Workspace File 3 In the Import Export Utility window click Open then browse to the location to which you unzipped the workspace file and select ABI SNPLEX Workspace imp Import Export Utility m E EL S New Open Save Export Import Close Workspace Import File Workspace ABI SNPLEX_Workspace imp E L Deck Layouts a Deck Layouts Framing Toole t 4 ABI SNFLEX NC 2 7 Labware Labware Liquid Types io b AxyoenS6 5 ul Barrier Pipetting Templates 4 snplex_Axyoen_96_DiaBot_HP e Pod Settings snplex OLA ABI 384 in_ACME Adapter E Techniques snplex PCR amp BI 384 m ALME Adapter w Tips snplex VwR 96 PP 15pos amp LP ECJ Well Pattems snplex VR 36 PP TpasALP a Liquid Types snplex AssayMix enplex DINASample amp nplex Exodi snplex OQL amp Rxn snplex PCR E Pipetting Templates Default Template ie Low Volume EI Techniques POR snplex_AssayMix_SuL amp nplex amp ssayMix buL enplex DMA Sul snplex ExoDil 15uL enplex Exolim 5uL snplex_OLARixn_ 2 08uL o 4 snplex PCRMis 7 92uL Tips i AxyPS0 Bamer tettet 9 Drag and drop workspace elements to import or export 4 Click Open to display the SNPlex System workspace in the Import File pane 5 Select ABI SNPLEX Workspac
74. te Position Method 1c_wet Deck Layout 3 OLA1 to OLAA P1 to P4 DNA sample P18 SN es A SN Mix9 to Mix12 P5 to P8 OLA Mix10 Uncovered tip P9 to P12 es P14 box DNA Tip OLA Mix11 DNA Samples OLA Mix12 j o S 9 Click OK to continue the method 10 When the method pauses remove the four used tip boxes in positions P9 to P12 and the Assay Mix plates Mix9 to Mix12 from positions P5 to P8 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 47 Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Method 1c wet 11 Remove the tips corresponding to well positions G12 and H12 from four new tip boxes and place them and the plates as shown in the deck layout below box HSUHU ETE DNA Samples F18 E EE ES Plate Position Method 1c wet Deck Layout 4 OLA1 to OLAA P1 to P4 DNA sample P18 SN H E Mix13 to P5 to P8 Mix16 E Uncovered tip P9 to P12 L 5 L 12 Click OK to continue the method 13 After the method run is complete remove and discard the tip boxes and plates Mix13 to Mix16 and remove seal and store if necessary the DNA Samples plate 14 Remove seal and briefly centrifuge the OLA reaction plates OLA1 to OLA4 15 Proceed to Thermal Cycling the OLA Reactions Thermal Cycling Thermal cycle the OLA plates using the follo
75. tem 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 6 Diluting the Purified OLA Product amp Introduction _ Setting Up the A Biomek FX for Automating the SNPlex System Assay cov s 5 Performing OLA c Performing OLA 4 V Using Dry DNA 4 V Using Wet DNA Purifying OLA Exonuclease See page 60 Overview Diluting the Purified gt OLA Product Running Biomek FX Method 3 Dilution See page 61 Setting Up the PCR Reactions Notes SNPlex M Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 59 Chapter 6 Diluting the Purified OLA Product Overview Overview About this This chapter provides information on how to dilute SNPlex OLA reaction plates Chapter previously purified by exonuclease using the Biomek FX liquid handling instrument to dispense nuclease free water Where You Are In the SN Plex Design and order System Assay SNPlex ligation probes Workflow Design sample plate layout d POR T Run PCR Prepare hybridization plates Prepare gDNA PCR Laboratory and bind PCR product to plates OLA Laboratory Phosphorylate and Phosphorylate and 2 Z Add denaturant isolating ligate probes ligate probes 2 biotinylated strand on linkers and linkers and cim hybridization plate gDNA OLA
76. the OLA reaction is complete Proceed to Chapter 5 Purifying OLA Products Exonuclease 34 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide S VE BLU z i gt q a u lt gt Using Dry DNA e a Faaa u s A Pr et OTE A TR Performing OLA Chapter 4 Performing OLA Using Wet gDNA Introduction Setting Up the Biomek FX for Automating the SNPlex System Assay Performing OLA NV Using Wet DNA Purifying OLA Products Exonuclease Diluting the Purified OLA Product Setting Up the PCR Reactions Notes oee page 36 Overview Plate Layouts and Methods for the OLA Protocol Wet gDNA See page 37 Performing the OLA Reactions Biomek FX Methods 1a Wet or 1b Wet See page 40 or Performing the OLA See page 45 Reactions Biomek FX Method 1c Wet or Performing the OLA Reactions Biomek FX Method 1d_Wet See page 49 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 35 Chapter 4 Performing OLA Using Wet gDNA Overview Overview About This This chapter provides information about automating the OLA protocol with the Biomek Chapter FX with disposable tips The procedures in this chapter assume the use of a batch of four SNPlex OLA reaction plates and that
77. the gDNA samples have been quantified fragmented and diluted to a concentration of 18 5 ng uL before beginning this procedure If you are using dry gDNA refer to Chapter 3 Performing OLA Using Dry gDNA Where You Are In the SN Plex Design and order System Assay SNPlex ligation probes Workflow I s Design sample plate layout T Run PCR we dud D fe fe E E l ed m q 5 Prepare hybridization plates Oo Prepare gDNA a and bind PCR product to plates 2g E Phosphorylate and Phosphorylate and Z Add denaturant isolating 6 ligate probes ligate probes o biolinylated strand on S linkers and linkers and i hybridization plate gDNA OLA dry gDNA OLA wet oa gt 3 l E Hybridize ZipChute M probes Q Purify OLA products E 2 Exonuclease uc E a l Elute ZipChute probes e Dilute purified OLA product 3 S Q gt O Prepare sample plates 2 for electrophoresis Prepare PCR reactions Amplification Create results groups and plate records Load and run sample plates Analyze data in GeneMapper software v3 7 36 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA ee Plate Layouts and Methods for the OLA Protocol Wet gDNA Plate Layouts and Methods for the OLA Protocol Wet gDNA You can select from four Biomek FX methods dependi
78. the tips corresponding to well positions G12 and H12 from every tip box Method 1a wet or I 1b wet DIES a E N OOOOOOOG6 OOOOOOO COOCOO OOOOOOG 0 0 0 0 0 0 0 0 00000000 000000 o0 09 00 Remove tips from wells G12 and H12 2 Place the uncovered tip boxes and plates on the deck as shown in the following figure placing an ACME adapter underneath each OLA plate Plate Position Methods 1a wet and 1b wet Deck Layout 1 OLA1 to OLAA P1 to PA Mix1 to Mix4 P5 to P8 Uncovered tip P9 to P12 box 3 Start the method using either la SNPLEXv5 OLA PN S4 W bmt for Method la wet OT e Jb SNPLEXv5 OLA P4 SN W bmt for Method Ib wet When the method pauses verify that the deck layout corresponds to the figure above and select OK to continue 42 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Methods 1a wet or 1b wet 4 When the method pauses remove and discard the tip boxes and plates Mix1 to Mix4 Remove the tips corresponding to well positons G12 and H12 from eight new uncovered tip boxes and place them the DNA sample plates on the deck as shown in the deck layout below Plate Position Methods 1a wet and 1b wet Deck Layout 2 and 3 OLA1 to OLAA P1 to PA DNA1 to DNA4 P5 to P8 Uncovered tip P9 to P16 box TL1
79. tware to correctly analyze the data Each run must include an allellic ladder control DNA and no template control NTC For additional information about sample plate layout refer to the SNP lex Genotyping System 46 plex General Automation Getting Started Guide Note that different robotics manufacturers have differing conventions for the order and location of plate quadrants Be cognizant of these differences when programming robotics 8 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 2 setting Up the Biomek FX for Automating the SNPlex System Assay Introduction See page 10 Setting Up the Overview Biomek FX for Automating the SNPlex System Assay Creating and Naming See page 12 the Deck Layout ES Performing OLA A Performing OLA X Using Dry DNA C NX Using Wet DNA Framing the Deck See page 13 Importing the See page 13 Purifying OLA Workspace File iu cssase cess Products Exonuclease Copying the Method Files See page 15 c Diluting the Purified ame OLA Product Reviewing the Supplied See page 16 Methods Setting Up the PCR Reactions Notes SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 9 Overview Overview Chapter 2 Setting Up the Biomek FX for Automating the SNPlex System Assay A
80. ur choice then click Search Find the document of interest right click the document title then select any of the following Open To view the document Print Target To print the document Save Target As To download a PDF version of the document to a destination that you choose Chemical Safety To minimize the hazards of chemicals Read and understand the Material Safety Data Sheets MSDS provided by the Guidelines chemical manufacturer before you store handle or work with any chemicals or hazardous materials See About MSDSs on page vii Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the MSDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the MSDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended on the MSDS Comply with all local state provincial or national laws and regulations related to chemical storage handling and disposal viii SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Safety and EMC Compliance Information Chemical Waste Safety Chemical Waste Safety Chemical Waste
81. uring this process coordinates of each position on the deck are established enabling the instrument to move objects to and from various locations on the deck precisely IMPORTANT Framing the deck ensures the correct transfer of materials which is essential to proper method operation Do not use the Biomek FX instrument unless you have completed the deck framing process Refer to the Biomek FX documentation for more information about this process Importing the Workspace File Downloading the Zip Archive Importing the Workspace File Note Because the zip archive contains both the workspace and method files you need only download it once 1 Go to http www appliedbiosystems com Click Support at the top of the page On the Support page click Software Downloads 2 3 4 From the Select Product Software menu select SNPlex Genotyping System 5 From the Software Type menu select Main Page 6 Download the appropriate zip archive for your robot IMPORTANT If you use the Biomek FX instrument for tasks other than automating the SNPlex System assay back up your workspace before importing the SNPlex System workspace file Workspace parameters for the SNPlex System assay may be different than those for other configurations and may overwrite those parameters Refer to the Biomek FX software documentation for information on backing up a workspace 1 Start the Biomek FX software 2 Select Tools gt Import Expor
82. wing conditions the OLA Reactions Step Step Type Temperature C Time 1 HOLD 48 30 min 2 HOLD 90 20 min 3 25 cycles 94 15 sec 60 30 sec 51 3 ramp 30 sec 4 HOLD 99 10 min 5 HOLD 4 Next Steps At this point the OLA reaction is complete Proceed to Chapter 5 Purifying OLA Products Exonuclease 48 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Method 1d wet Performing the OLA Reactions Method 1d_wet About The Method 1d wet was developed for setups that have 1472 gDNA samples and 1 ligation Method probe pool and uses four deck layouts Assay Mix is added to all OLA plates then DNA samples from source plates DNA1 to DNA4 are transferred to plate OLAT e DNA samples from source plates DNA5 to DNAS are transferred to plate OLA2 e DNA samples from source plates DNA9 to DNA12 are transferred to plate OLA3 e DNA samples from source plates DNA13 to DNA16 are transferred to OLA4 Preparing the 1 Label a skirted 96 well PCR plate Assay Mix Reagents 2 Label 16 wet DNA source plates DNAI to DNA16 For information on setting up DNA source plates see Arraying gDNA in 96 Well Plates on page 37 3 Prepare the Assay Mix in a 15 mL centrifuge tube and mix thoroughly Automated Manual Automation Losses ALSIEME T
83. x 4 7 15 7 3 6 38 1 SNPlex dATP 4 7 15 7 3 6 38 1 SNPlex Probe Pool 500 nM 9 4 31 3 7 3 76 2 Total 282 0 940 0 218 0 2286 0 94 tips in source plate x 10uL per tip Transfer losses for transferring bulk mixture into 96 well source plate Use the volumes in this column to prepare the reagents Refer to the SNPIex Genotyping System 48 plex User Guide for per reaction volumes Volumes given in this table include allowances for dead volume and excess volume for pipetting losses For more information about calculating dead volumes refer to the SNPIex Genotyping System 48 plex General Automation Getting Started Guide Qo 6 Add 22 uL of the Assay Mix into each well of a skirted 96 well PCR plate excluding wells G12 and H12 then centrifuge the plate briefly to collect the contents at the bottom of the wells Mix 1 deoecc0000000 00000000000 22 uL 900000000000 00000000000 000000000000 Y 00000000000 90000000000C 000000000000 Leave wells G12 and H12 empty f Label four 384 well ABI clear optical reaction plates OLA1 to OLA4 For information on the layout of wet gDNA plates see Method la wet or lb wet on page 37 SNPlex Genotyping System 48 plex Automating OLA Using the Biomek FX Getting Started Guide 41 Chapter 4 Performing OLA Using Wet gDNA Performing the OLA Reactions Biomek FX Methods 1a wet or 1b wet Running 1 Remove

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