DNA IQ Casework Pro Kit for Maxwell 16 Technical Manual #TM332
Contents
1. if necessary DNA Extraction from Epithelial Fraction 1 To an epithelial fraction of up to 400ul add 200ul of Lysis Buffer 2 Close the lid of the tube and vortex the sample for 5 10 seconds 3 The sample is now ready for automated DNA extraction using the Maxwell 16 LEV Instrument Proceed to Section 5 for cartridge preparation and instrument setup Note Store preprocessed sample at room temperature 15 30 C overnight if necessary Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 7 Promega 5 Maxwell 16 Automated DNA Purification Protocol 5 A Preparation of Samples for Maxwell 16 LEV Cartridges 1 Change gloves before handling cartridges LEV Plungers and Elution Tubes Place the cartridges to be used in the Maxwell 16 LEV Cartridge Rack Cat AS1251 Place each cartridge in the rack with the label side facing away from the Elution Tubes Press down on the cartridge to snap it into position Carefully peel back the seal so that all plastic comes off the top of the cartridge Ensure that all sealing tape and any residual adhesive are removed before placing cartridges in the instrument Notes 1 If you are processing fewer than 16 samples center the cartridges on the platform 2 Clean sample or reagent spills on any part of the Maxwell 16 LE2. IQ Spin Basket with forceps being sure to orient the swab or fabric toward the bottom of the spin basket Transfer the lysate from the incubation tube to the spin basket and close the tube 6 Centrifuge at room temperature for 2 minutes at maximum speed in a microcentrifuge Carefully remove the DNA IQ Spin Basket Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 5 Promega 4 A Samples on a Solid Support continued 7 Add 200ul of Lysis Buffer to the tube containing extract 8 Close the lid of the tube and vortex the sample for 5 10 seconds 9 The sample is now ready for automated DNA extraction using the Maxwell 16 LEV Instrument Proceed to Section 5 for cartridge preparation and instrument setup Note Store preprocessed sample at room temperature 15 30 C overnight if necessary 4 B Liquid Samples This preprocessing protocol results in optimal lysis from samples in an aqueous solution using a proteinase K treatment Materials to Be Supplied by the User e 56 C heat block or water bath ClickFit Microtube 1 5ml Cat V4741 e Casework Extraction Kit Cat DC6745 e Aerosol resistant pipette tips Note Using the ClickFit Microtube prevents the lid from opening during heated incubation Preparation of Stock Proteinase K Solution
3. IQ Spin Baskets Cat V1221 e Casework Extraction Kit Cat DC6745 e Aerosol resistant pipette tips Note We recommend using the ClickFit Microtube with DNA IQ Spin Baskets to ensure optimal fit and ease of use In addition the ClickFit Microtube prevents the lid from opening during heated incubation Preparation of Stock Proteinase K Solution for Sample Preprocessing Add 556ul of Nuclease Free Water to one tube of lyophilized Proteinase K and gently invert to dissolve The final concentration of Proteinase K will be 18mg ml Store Proteinase K Solution at 20 C Extraction of Samples on a Solid Support 1 Place solid substrate e g fabric or swab head at the bottom of a labeled ClickFit Microtube 1 5ml 2 Add the final volume of each reagent listed in Table 1 to a sterile tube Table 1 Extraction Mix for Solid Support Samples Volume Per Number of Extraction Mix Component Extraction x Extractions Final Volume Casework Extraction Buffer 38 XxX s Proteinase K 18mg ml 10u x 1 Thioglycerol Aul x Total Reaction Volume 400l x Note 1 Thioglycerol is viscous Pipet slowly 3 Briefly vortex the Extraction Mix and dispense 400u1 to each ClickFit Microtube containing solid substrate 4 Close the tube lid vortex sample at high speed for 5 seconds and incubate the sample at 56 C for 30 minutes 5 Place a DNA IQ Spin Basket into a labeled ClickFit Microtube 1 5ml Transfer the sample to the DNA
4. LEV and the LEV hardware is present Press Run to continue 3 Enter user and PIN if this option is enabled 4 At the Protocols screen select Casework 5 On the next screen verify that the correct method and user were chosen Select Run Stop to continue 6 Open the door when prompted on the screen then select Run Stop A Warning Pinch point hazard Follow on screen instructions for bar code reader input if this option is enabled 8 Transfer the Maxwell 16 LEV Cartridge Rack containing the prepared cartridges to the Maxwell 16 Instrument platform Ensure that the rack is placed in the Maxwell 16 Instrument with the Elution Tubes closest to the door The rack will only fit in the instrument in this orientation If you have difficulty fitting the rack on the platform check that the rack is in the correct orientation Ensure the rack is level on the instrument platform Note Hold the Maxwell 16 LEV Cartridge Rack by the sides to avoid dislodging cartridges from the rack 9 Verify that samples were added to well 1 of the cartridges cartridges in the rack are loaded on the instrument Elution Tubes are present with 501 of Elution Buffer and LEV Plungers are in well 8 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 11 Prom
5. for Sample Preprocessing Add 556ul of Nuclease Free Water to one tube of lyophilized Proteinase K and gently invert to dissolve The final concentration of Proteinase K will be 18mg ml Store Proteinase K Solution at 20 C Extraction of Liquid Samples 1 Pipet liquid sample into the bottom of a labeled ClickFit Microtube 1 5ml and add 10ul of Proteinase K Solution 4ul of 1 Thioglycerol and Casework Extraction Buffer to a total volume of 400uI Do not exceed a final volume of 400uI Note 1 Thioglycerol is viscous Pipet slowly 2 Close the tube lid vortex sample at high speed for 5 seconds and incubate the sample at 56 C for 30 minutes 3 Add 200uIl of Lysis Buffer to each sample 4 Close the lid of the tube and vortex the sample for 5 10 seconds 5 The sample is now ready for automated DNA extraction using the Maxwell 16 LEV Instrument Proceed to Section 5 for cartridge preparation and instrument setup Note Store preprocessed sample at room temperature 15 30 C overnight if necessary Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 6 Revised 1 14 Promega 4 C Differential Extraction Samples Differential extraction is the classic method to lysis of epithelial cells using proteinase K in the absence of DTT to prevent the lysis of spermatozoa This proce
6. maximize the amount of DNA purified from forensic casework materials use the protocol appropriate for your sample type Protocols include 1 Samples on a Solid Support Section 4 A 2 Liquid Samples Section 4 B 3 Differential Extraction Samples Section 4 C All protocols involve a Proteinase K treatment which is required to maximize recovery and yield from a variety of sample types including small amounts of sample on a solid matrix such as a swab or fabric DNA samples extracted using a Proteinase K treatment generally exhibit better locus to locus balance in downstream STR analysis Note These preprocessing protocols use the Casework Extraction Buffer included in the Casework Extraction Kit Cat DC6745 for the Proteinase K digestion If another proteinase K digestion buffer is used for comparison the concentration of SDS must be below 0 5 or a precipitate may form when the Lysis Buffer is added Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 4 Revised 1 14 Promega 4 A Samples on a Solid Support This preprocessing protocol allows optimal DNA extraction from samples on a solid support such as swabs or fabric using a Proteinase K incubation Materials to Be Supplied by the User e 56 C heat block or water bath e ClickFit Microtube 1 5ml Cat V4741 e DNA
7. 4 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 2 Revised 1 14 Promega 2 Product Components and Storage Conditions Product Size Cat DNA IQ Casework Pro Kit for Maxwell 16 48 preps AS1240 Not For Medical Diagnostic Use Sufficient for 48 automated isolations from forensic casework samples Includes 48 Maxwell 16 LEV Cartridges MCB 50 LEV Plungers 50 Elution Tubes 0 5ml 20m Elution Buffer 32ml Lysis Buffer Purchase Separately Product Size Cat Casework Extraction Kit 100 reactions DC6745 Not For Medical Diagnostic Use Sufficient for 100 reactions of 400ul each Includes 50ml Casework Extraction Buffer e 2x 10mg Proteinase K 900ul 1 Thioglycerol e 1 25ml Nuclease Free Water Storage Conditions Store the DNA IQ Casework Pro Kit for Maxwell 16 and Casework Extraction Buffer at 15 30 C Store 1 Thioglycerol at 2 10 C Store Proteinase K at 30 to 10 C Store Nuclease Free Water below 30 C Safety Information The reagent cartridges contain flammable substances ethanol and isopropanol and the harmful substance guanidine thiocyanate 1 Thioglycerol is a toxic substance Well Contents User Adds 1 Lysis Buffer Sample 2 DNA IQ Resin 3 Lysis Buffer 4 Wash Buffer 5 Wash Buffer 6 Wash Buffer 7 Empty 8592TB 8 Empty Plunger Figure 2 Maxwell 16 LEV Cartridge Promega Corporation 2800 Woods Hollow Road Madison W
8. I 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 3 Promega 3 Maxwell 16 Instrument Hardware and Firmware Setup The Maxwell 16 Instrument Cat AS2000 or Maxwell 16 Forensic Instrument Cat AS3060 configured for low volume elution LEV is required for use with the DNA IO Casework Pro Kit for Maxwell 16 Users with a Maxwell 16 Instrument configured for standard elution volume SEV need to reconfigure the instrument using the Maxwell 16 LEV Hardware Kit Cat AS1250 Reconfiguring the instrument is easy For instructions to set up the instrument please refer to the Maxwell 16 Instrument Technical Manual TM295 for Cat AS2000 or the Maxwell 16 Forensic Instrument Technical Manual TM321 for Cat AS3060 The first time that the Maxwell 16 Instrument is powered up a series of user prompts will appear on the Navigation LCD The DNA IQ Casework Pro Kit for Maxwell 16 is intended for use with the LEV low elution volume settings and the Forensic method on the instrument Once the Forensic method is set up on the instrument all subsequent power ups of the instrument will automatically default to these settings 4 Sample Preprocessing Depending on the sample type there are different preprocessing steps that must be performed prior to running the automated method for the Maxwell 16 Instrument To
9. T 5g DIT 32 4ml Nuclease Free Water Add Nuclease Free Water to DTT and swirl briefly to dissolve Dispense 1M DTT into smaller aliquots that reflect usage and store at 20 C Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 14 Revised 1 14 Promega 9 Related Products Product Size Cat Maxwell 16 LEV Hardware Kit leach AS1250 Maxwell 16 LEV Cartridge Rack leach AS1251 DNA IQ Reference Sample Kit for Maxwell 16 48 preps AS1040 Differex System 50 samples DC6801 200 samples DC6800 Proteinase K 100mg V3021 Maxwell 16 LEV Magnet leach AS51261 Maxwell 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor leach SP1070 MagneSphere Technology Magnetic Separation Stand twelve position 1 each 25341 ClickFit Microtube 1 5ml 1 000 pack V4741 U S Pat Nos 6 027 945 6 368 800 and 6 673 631 European Pat No 1 204 741 and Japanese Pat No 4425513 2010 2014 Promega Corporation All Rights Reserved MagneSphere and Maxwell are registered trademarks of Promega Corporation Differex and DNA IQ are trademarks of Promega Corporation Products may be covered by pending or issued patents or may have certain limitations Please visit our Web site for more information All prices and specifications are subject to change without prior notice Product claims are su
10. TECHNICAL MANUAL DNA IQ Casework Pro Kit for Maxwell 16 Instructions for use of Products AS1240 and DC6745 Pig Mega Promega DNA IQ Casework Pro Kit for Maxwell 16 All technical literature is available on the Internet at www promega com protocols Please visit the web site to verify that you are using the most current version of this Technical Manual Please contact Promega Technical Services if you have questions on use of this system E mail techserv promega com L NCSC BAI case E AE ceo anes E cscs vs tess ects eee 1 2 Product Components and Storage Conditions oe ceeeeeteeeeeeeneees 3 3 Maxwell 16 Instrument Hardware and Firmware Setup 00 4 4 Sample Pre COC CS SUING enurrpeinsoenaiisn EA A Samples ona Solid Go b 0 01y aeemeent mee tae Cente ee eee eee eee eee ee 5 Pe e n te te te one tener E ee 6 C Differential Extraction Samples csesssessceceseceeeeesseeeeseeeeeeneeneeneeneeees 7 5 Maxwell 16 Automated DNA Purification Protocol eee 8 A Preparation of Samples for Maxwell 16 LEV Cartridges oes 8 B Setup for AS52000 Maxwell 10 TAS tt WiC A ts ass 9 oe cectniyscrsersmereteneteerewencte 9 C Setup for AS53060 Maxwell 16 Forensic Instrument 0 eeeeeee 11 D otorne Hated DN Aseria n n inaani 12 Ge OF 0 6s C22 noo E soea T A 13 7 Extraction Buffer Options ccscssssssscssssssscsssssscsssscsscsesecseesscnsesenseseneees 14 8 Composition of So
11. V Cartridge Rack with a detergent water solution followed by 70 ethanol then water Do not use bleach on any instrument parts 2 Rotate the cartridge rack 180 degrees such that well 1 is closest to you Add sample lysate to well 1 Repeat for all samples Note The order of samples is now 16 1 left to right Ensure your samples are added in the correct order 3 Change gloves and rotate the rack 180 degrees again so the well 1 is furthest from you and place an LEV Plunger in well 8 of each cartridge Well 8 is the well closest to the Elution Tube Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 8 Revised 1 14 Promega 4 Place Elution Tubes in the front of the Maxwell 16 LEV Cartridge Rack Add 50u1 of Elution Buffer to the bottom of each Elution Tube Notes 1 Ensure that the Elution Buffer is in the bottom of the tube If Elution Buffer is on the side of the tube the elution may be suboptimal 2 Use only the Elution Tubes provided with the kit other tubes may not work with the Maxwell 16 Instrument 3 Use only the Elution Buffer supplied with the DNA IQ Casework Pro Kit for Maxwell 16 other buffers can negatively affect downstream analysis e g poor quality STR profiles 5 Proceed to Section 5 B for Cat AS2000 instruments or Section 5 C for C
12. at AS3060 instruments 5 B Setup for AS2000 Maxwell 16 Instruments Refer to the Maxwell 16 Instrument Operating Manual TM295 for more detailed information To run the Casework protocol you must have Maxwell 16 firmware version 4 0 or higher installed on your instrument 1 Turn on the Maxwell 16 Instrument The instrument will power up display the firmware version number proceed through a self check and home all moving parts 2 Verify that the instrument settings indicate an LEV hardware configuration and Fnsc operational mode setting 3 Select Run on the Menu screen and press the Run Stop button to start the method 4 Select DNA on the Menu screen then select OK at the Verification screen 5 Open the door when prompted to do so on the screen Press the Run Stop button to extend the platform A Warning Pinch point hazard Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 9 Promega 5 B Setup for AS2000 Maxwell 16 Instruments continued 6 Transfer the Maxwell 16 LEV Cartridge Rack containing the prepared cartridges to the Maxwell 16 Instrument platform Ensure that the rack is placed in the Maxwell 16 Instrument with the Elution Tubes closest to the door The rack will only fit in the instrument
13. bject to change Please contact Promega Technical Services or access the Promega online catalog for the most up to date information on Promega products Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 15
14. ega 10 Press the Run Stop button The platform will retract Close the door Warning Pinch point hazard The Maxwell 16 Forensic Instrument will immediately begin the purification run The screen will display the steps performed and the approximate time remaining in the run Notes 1 Pressing the Run Stop button or opening the door will pause the run 2 If the run is abandoned before completion the instrument will wash the particles off the plungers and eject the plungers into well 8 of the cartridge However the sample DNA will remain on the particles and can be recovered easily either by replacing the plungers in well 8 and rerunning the purification or by removing the well contents and manually finishing the isolation process as instructed in the DNA IQ System Small Sample Casework Protocol Technical Bulletin TB296 11 When the automated purification run is complete follow instructions on the screen for data transfer For detailed instructions refer to the Maxwell 16 Forensic Instrument Technical Manual TM321 and Maxwell Sample Track Software Technical Manual TM314 End of Run 12 Follow on screen instructions at the end of the method to open the door Verify that plungers are located in well 8 of the cartridges If plungers are not removed from the magnetic plunger bar push them down gently by hand to remove them 13 Press the Run Stop button to extend the platform out of the instrument 14 Remove
15. in this orientation If you have difficulty fitting the rack on the platform check that the rack is in the correct orientation Ensure that the cartridge rack is level on the instrument platform Note Hold the Maxwell 16 LEV Cartridge Rack by the sides to avoid dislodging cartridges from the rack 7 Verify that samples were added to well 1 of the cartridges Elution Tubes are present with 50ul of Elution Buffer and LEV Plungers are in well 8 8 Press the Run Stop button The platform will retract Close the door A Warning Pinch point hazard 9 The Maxwell 16 Instrument will immediately begin the purification run The screen will display the steps performed and the approximate time remaining in the run Notes 1 Pressing the Run Stop button or opening the door will pause the run 2 If the run is abandoned before completion the instrument will wash the particles off the plungers and eject the plungers into well 8 of the cartridges However the sample DNA will remain on the particles and can be easily recovered either by replacing the plungers in well 8 and rerunning the purification or by removing the well contents and manually finishing the isolation process as instructed in the DNA IQ System Small Sample Casework Protocol Technical Bulletin TB296 10 When the automated purification run is complete the LCD screen will display a message that the method has ended End of Run 11 Follow on screen instructi
16. lution sssssessssessssssressssrresssttessstteesssressssrressrresssreeesss 14 9 Related Products cea ctaecee tc tee eecaceececaa pe eaeaaatessaeep te aaeeeyetacas a tareerenceetes 15 1 Description The DNA IQ Casework Pro Kit for Maxwell 16 is used with the Maxwell 16 Instrument Cat AS52000 Cat AS3060 configured for low elution volume LEV and is specifically designed for optimal DNA extraction from forensic casework samples These samples include blood stains semen stains hair cigarette butts tissue samples and touch DNA samples regularly encountered in forensic DNA analysis The Casework Extraction Kit was developed to improve DNA extraction efficiency from a broad panel of sample types Figure 1 This protocol describes the use of the Casework Extraction Kit Cat DC6745 for preprocessing samples before DNA extraction with the DNA IQ Casework Pro Kit for Maxwell 16 Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 1 Promega 1 Description continued E Incubation Buffer 7 Casework Extraction Buffer Estimated Yield ng Blood on Blood on Saliva on blue denim cotton shirt envelope 10341MA Figure 1 DNA was extracted from mock casework sample types using the Incubation Buffer from the Tissue and Hair Extractio
17. n Kit Cat DC6740 or Casework Extraction Buffer and isolated using the DNA IQ Casework Pro Kit for Maxwell 16 Extraction from certain sample types e g sperm cells traditionally requires the use of dithiothreitol DTT as part of the extraction method The Casework Extraction Kit includes 1 Thioglycerol which unlike DTT can be stored at 2 10 C and provides extraction performance comparable to that with DTT This Technical Manual provides instructions to use 1 Thioglycerol for sample preprocessing The Maxwell 16 LEV Instrument is supplied with preprogrammed DNA purification procedures uses prefilled reagent cartridges and elutes DNA samples in small volumes The instrument can process up to 16 samples in approximately 30 minutes The DNA IQ Casework Pro Kit for Maxwell 16 uses the DNA IQ Resin to purify DNA maximizing DNA yield and purity for use in STR analysis The Maxwell 16 Instrument is a magnetic particle handling instrument that efficiently transports the DNA IQ Resin through purification reagents in prefilled cartridges Figure 2 mixing the resin with the reagents during processing The paramagnetic particle based methodology avoids common problems experienced with other automated systems such as clogged tips or partial reagent transfers which can lead to suboptimal DNA purification Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 27
18. nsions Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Printed in USA Part TM332 Revised 1 14 Page 13 Promega 6 Troubleshooting continued Symptoms Causes and Comments Resin carryover during elution A small amount of resin was visible in the Elution Tube The presence of resin particles will not affect the final DNA concentration or downstream applications If desired an additional resin capture step may be performed using the Maxwell 16 LEV Magnet Cat AS1261 or 0 5ml MagneSphere Technology Magnetic Separation Stand Cat Z5341 Use the Maxwell 16 High Strength LEV Magnetic Rod and Plunger Bar Adaptor Cat SP1070 to significantly improve the recovery of resin particles at each step and reduce the amount of resin in the Elution Tube 7 Extraction Buffer Options Additional extraction buffers are compatible with the DNA IQ Casework Pro Kit for Maxwell 16 The Casework Extraction Buffer provided as part of the Casework Extraction Kit Cat DC6745 is recommended but different laboratories have reported success with Bone Incubation Buffer Bone Incubation Buffer 10mM Tris 100mM NaCl 50mM EDTA 0 5 SDS Note Some end users prefer to process reference samples in heated DNA IQ Lysis Buffer We do not recommend this method for casework samples 8 Composition of Solution 1M DT
19. ons at the end of the method to open the door Verify that plungers are located in well 8 of the cartridges If plungers are not removed from the magnetic plunger bar push them down gently by hand to remove them 12 Press the Run Stop button to extend the platform out of the instrument 13 Remove the Maxwell 16 LEV Cartridge Rack from the instrument Remove Elution Tubes containing DNA and close the tubes Note Some paramagnetic particles may be present in the Elution Tube and can be removed by capturing particles using the Maxwell 16 LEV Magnet Cat AS1261 or 0 5m MagneSphere Technology Magnetic Separation Stand Cat Z5341 and transferring the DNA to a new tube Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 10 Revised 1 14 Promega 14 Remove cartridges and plungers from the Maxwell 16 LEV Cartridge Rack and discard as hazardous waste Do not reuse reagent cartridges LEV Plungers or Elution Tubes 5 C Setup for AS3060 Maxwell 16 Forensic Instruments Refer to the Maxwell 16 Forensic Instrument Technical Manual TM321 for detailed information 1 Turn on the Maxwell 16 Forensic Instrument The instrument will power up display the firmware version number proceed through a self check and home all moving parts 2 Verify that the Home screen indicates
20. ss separates a forensic sample into two fractions sperm and epithelial The Differex System Cat DC6801 DC6800 can be used to quickly and efficiently separate male and female sample fractions See the Differex System Technical Manual TM331 for a protocol to separate sperm and epithelial fractions available at www promega com protocols Sperm Fraction Protocol Materials to Be Supplied by the User e ClickFit Microtube 1 5ml Cat V4741 Nuclease Free Water Cat P1193 e Aerosol resistant pipette tips Note Using the ClickFit Microtube prevents the lid from opening during heated incubation Preparation of Lysis Buffer 1 Determine the total volume of prepared Lysis Buffer to be used 2 Add 1p of 1 Thioglycerol for every 400p1 of Lysis Buffer Note 1 Thioglycerol is viscous Pipet slowly Alternatively add 1pl of 1M DTT for every 100pl of Lysis Buffer See Section 8 Composition of Solution for preparation instructions DNA Extraction from Sperm Fraction 1 Add 400ul of prepared Lysis Buffer to the sperm fraction generally a pellet or liquid volume less than 100ul in a labeled ClickFit Microtube 1 5ml 2 Close the lid of the tube and vortex the sample for 5 10 seconds 3 The sample is now ready for automated DNA extraction using the Maxwell 16 Instrument Proceed to Section 5 for cartridge preparation and instrument setup Note Store preprocessed sample at room temperature 15 30 C overnight
21. ssing to increase yield Too much sample was processed DNA isolation using the Maxwell 16 LEV Cartridge is most efficient when there is 8001 in well 1 Larger volumes can be processed but may reduce isolation efficiency Insufficient Lysis Buffer Dispense an equal volume of Lysis Buffer to sample volume during the binding step in well 1 A minimum of 200ul of Lysis Buffer should be dispensed into the Maxwell 16 LEV Cartridge Insufficient lysis of sperm cells after differential extraction Sperm samples typically require extraction with proteinase K Instrument calibration error Verify nothing is physically blocking the movement of the platform plunger bar or magnetic rod assembly Turn the machine off then on to cycle the power The instrument will rehome itself If the calibration error occurs again after power cycling contact Promega for service Turn the machine off then on to cycle the power After cycling power run a Demo method without any cartridges in the machine If another calibration error occurs during the Demo run contact Promega for service Ensure the Maxwell 16 LEV Hardware Kit Cat AS1250 is installed The cartridges were not completely seated on the platform Ensure the cartridges are pressed firmly into place Incorrect elution tube was used Use only the 0 5ml Elution Tube provided with the DNA IQ Casework Pro Kit for Maxwell 16 Other tubes may have different dime
22. the Maxwell 16 LEV Cartridge Rack from the instrument Remove Elution Tubes containing DNA and cap the tubes Note Some paramagnetic particles may be present in the Elution Tube and can be removed by capturing particles using the Maxwell 16 LEV Magnet Cat AS1261 or 0 5ml MagneSphere Technology Magnetic Separation Stand Cat Z5341 and transferring the DNA to a new tube 15 Remove cartridges and plungers from the cartridge rack and discard as hazardous waste Do not reuse reagent cartridges LEV Plungers or Elution Tubes Note Ensure samples are removed before the UV light treatment to avoid damage to the nucleic acid 5 D Storing Eluted DNA If DNA is not analyzed immediately store the eluted DNA on ice or at 4 C for up to 24 hours For longer term storage consult laboratory guidelines Freezing samples at 20 C or 70 C has been shown to preserve DNA for longer periods of time Promega Corporation 2800 Woods Hollow Road Madison WI 53711 5399 USA Toll Free in USA 800 356 9526 Phone 608 274 4330 Fax 608 277 2516 www promega com Part TM332 Printed in USA Page 12 Revised 1 14 Promega 6 Troubleshooting For questions not addressed here please contact your local Promega Branch Office or Distributor Contact information available at www promega com E mail techserv promega com Symptoms Causes and Comments Low DNA yield Insufficient sample was processed Add more starting material for preproce
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