ABI PRISM 7700 - Thermo Fisher Scientific
Contents
1. Invitrogen life technologies Protocols LSS aaa LUX Primers Instrument Protocols For Using LUX Primers on the ABI PRISM 7700 Roche LightCycler Bio Rad iCycler ABI PRISM 7000 Corbett Rotor Gene 3000 Cepheid Smart Cycler DNA Engine Opticon 2 Stratagene Mx3000P ABI PRISM 7900 Version B 17 March 2003 25 0696 Instrument Protocols and Settings for LUX Primers TM Specific protocols have been developed for using LUX Primers on real time qPCR instruments These protocols all use Platinum Quantitative PCR SuperMix UDG available from Invitrogen Cat nos 11730 017 and 11730 025 For real time quantitative RT PCR we recommend the SuperScript III Platinum One Step qRT PCR Kit Cat nos 11732 020 and 11732 088 See the kit manual for a protocol using LUX Primers Protocol Page ABPPRISM IDOL edu ur M EE DE PM M EE LM UE LN LE EE 2 Roche LightGycler 22 5 5 aeu eeu ee lepus ded se epu a TE 4 Bio Rad iCyeler aucta test acitaqi t besitirqitmeitheqteedliiuu tend eet rte tiet 6 ABPPRSISNM SZUD G ies edet ate ie eka et trate Peli alse ate aa nd ctl tld tela Ue Ele LAT 8 Corbett Rotor Gene 30007 cec ct hac Eme ei fa Hg ha LE D LII i LE rU 10 Ceblield Smart Cycler oss dtetmdadeadatu adulatio A E 12 DNA Engine Opticon 23 28 26 eau Gael eee eae 14 Stratagene MX3000P oid a TE PE ON OE nios nist t desta 16 ABI PRISM A DOOUE Cote en a en ne en ee ee ne E 18 Trademark Information2. Action Temp C Time Cycles UDG reaction 50 2 min 1 UDG inactivation template denaturation 95 2 min 1 Denaturation 95 15 sec 40 50 Hybridization Elongation 60 30 sec Hold 60 30 sec Melting curve analysis select Dissociation Curve checkbox 60 Uracil DNA glycosylase mediated carry over prevention Continued on the next page Cycling Program continued Alternative Action Temp C Time Cycles UDG reaction 50 2 min 1 UDG inactivation template denaturation 95 2 min 1 Denaturation 95 15 sec Hybridization 55 30 sec 40 50 Elongation 72 30 sec Hold 60 30 sec Melting curve analysis select Dissociation Curve checkbox 60 Uracil DNA glycosylase mediated carry over prevention Protocol 1 Program the ABI PRISM 7000 as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 40 ul of the master mix to each tube strip well or well of the qPCR plate Add 10 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube strip plate 5 Gently mix and make sure that all components are at the bottom of the tube well Centr
3. 10 uM Tul 50 ul 200 nM Unlabeled primer 2 10 uM 1 ul 50 ul 200 nM MgCl 50 mM 3 pl 150 ul 3 mM ROX Reference Dye 50X 1 ul 50 ul 1X Autoclaved distilled water to 40 ul to 2000 ul 6 mM total Mg including MgCb in Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the ABI PRISM 7700 Recommended Action Temp C Time Cycles Ramp time Acquisition UDG reaction 50 2 min 1 Yes UDG inactivation template denaturation 95 2 min 1 Yes Denaturation 95 15 sec 40 50 Yes Hybridization Elongation 60 30 sec Yes Melting curve analysis 60 95 19 min In the ramp Alternative Action Temp C Time Cycles Ramp time Acquisition UDG reaction 50 2 min 1 Yes UDG inactivation template denaturation 95 2 min 1 Yes Denaturation 95 15 sec Yes Hybridization 55 30 sec 40 50 Yes Elongation 72 30 sec Yes Melting curve analysis 60 95 19 min In the ramp Uracil DNA glycosylase mediated carry over prevention Continued on the next page Protocol 1 Program the ABI PRISM 7700 as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 40 ul of the master mix to each tube strip well or well of the qPC
4. 100 reactions 500 reactions 100 reactions 500 reactions DNA Engine Opticon 2 The following real time quantitative PCR settings and protocol are designed for using LUX Primers on MJ Research s DNA Engine Opticon 2 See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 50 yl Monoplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 10uM 1u 50 ul 200 nM Unlabeled primer 10uM 1ul 50 ul 200 nM Autoclaved distilled water to 40 ul to 2000 pl Multiplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 1 10 uM 0 5 ul 25 ul 100 nM Unlabeled primer 1 10 uM 0 5 ul 25 ul 100 nM LUX labeled primer 2 10 uM Tul 50 ul 200 nM Unlabeled primer 2 10 uM 1 ul 50 ul 200 nM MgCl 50 mM 3 ul 150 ul 3 mM Autoclaved distilled water to 40 ul to 2000 ul 6 mM total Mg including MgCh in Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the DNA Engine Opticon 2 Recommended
5. Action Temp C Time Ramp time Acquisition 1 UDG reaction 50 2 min 2 C sec No 2 UDG inactivation template denaturation 95 2 min 2 C sec No 3 Denaturation 95 15 sec 2 C sec No 4 Hybridization Elongation 60 30 sec 2 C sec No 5 Plate read 0 2 C sec 1 sec plate read 6 Go to line 3 for 49 times No 7 Incubation 95 1 sec 2 C sec No 8 Melting curve 60 95 1 sec Read every 0 2 C 9 Incubation 25 30 min Uracil DNA glycosylase mediated carry over prevention Continued on the next page 14 Cycling Program continued Alternative Action Temp C Time Ramp time Acquisition 1 UDG reaction 50 2 min 2 C sec No 2 UDG inactivation template denaturation 95 2 min 2 C sec No 3 Denaturation 95 15 sec 2 C sec No 4 Hybridization 60 30 sec 2 C sec No 5 Elongation 72 30 sec 2 C sec No 6 Plate read 0 2 C sec 1 sec plate read 7 Go to line 3 for 49 times No 8 Incubation 95 1 sec 2 C sec No 9 Melting curve 60 95 1 sec Read every 0 2 C 10 Incubation 25 30 min Uracil DNA glycosylase mediated carry over prevention Protocol 1 Program the DNA Engine Opticon 2as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to redu
6. nM Unlabeled primer 1 10 uM 0 5 ul 25 ul 100 nM LUX labeled primer 2 10 uM 0 5 ul 25 ul 100 nM Unlabeled primer 2 10 uM 0 5 ul 25 ul 100 nM MgCb 50mM 3 pl 150 ul 3 mM ROX Reference Dye diluted 1 10 Dilute 1 10 lul 50 ul 1X Autoclaved distilled water to 40 ul to 2000 ul 6 mM total Mg including MgCl in Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the Mx3000P Action Temp C Time Cycles Acquisition UDG reaction 50 2 min 1 No UDG inactivation template denaturation 95 2 min 1 No Denaturation 95 15 sec No Hybridization 60 30 sec 40 50 Yes at the end Elongation 72 30 sec No Denaturation 95 1 min 30 sec at 55 C Melting curve analysis 55 95 and at 95 C Yes all Uracil DNA glycosylase mediated carry over prevention Continued on the next page 16 Protocol 1 o 6 Program the Mx3000P as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 40 ul of the master mix to each tube or plate well Add 10 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube p
7. sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors 3 For each reaction add 18 ul of the master mix to each tube use the 25 11 reaction tubes specific for the SmartCycler 4 Add 2 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube 5 Gently mix and make sure that all components are at the bottom of the tube Centrifuge briefly 6 Place reactions in the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument Follow the instructions in the Cepheid Smart Cycler user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument e Use the 25 ul reaction tubes specific for this instrument e Multiplex reactions are currently not recommended on the Smart Cycler e For each run keep all channels on e Use the default parameters for analysis Continued on the next page 12 Additional Products Product Platinum Quantitative PCR SuperMix UDG includes separate tubes of ROX Reference Dye and MgCl SuperScript III Platinum One Step qRT PCR Kit Catalog number 11730 017 11730 025 11732 020 11732 088 13 Size
8. 3 1 Denaturation 95 10 sec 40 50 At 50 C 2 Hybridization 50 30 sec yellow camera 3 Elongation 72 30 sec Cycle 4 Hold 95 1 min Cycle 5 Hold 55 1 min 0 5 C cycle after Cycle 6 Melting curve 55 10 sec 80 2 cycles Green camera Uracil DNA glycosylase mediated carry over prevention Protocol 1 Program the Bio Rad iCycler as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 40 ul of the master mix to each tube or plate well 4 Add 10 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube plate 5 Gently mix and make sure that all components are at the bottom of the tube well Centrifuge briefly if needed 6 Place reactions in the thermal cycler programmed as described above Collect and analyze the results Continued on the next page Tips and Recommendations for Programming the Instrument Follow the instructions in the Bio Rad iCycler user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument We recommend selecting Experimental Plate under Select well facto
9. 5 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions 19 e s Invitrogen life technologies United States Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Tel 1 760 603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 603 7229 Email tech serviceG invitrogen com European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF UK Tel Free Phone Orders 0800 269 210 Tel General Enquiries 0800 5345 5345 Fax 44 0 141 814 6287 Email eurotech invitrogen com International Offices Argentina 5411 4556 0844 Australia 1 800 331 627 Austria 0800 20 1087 Belgium 0800 14894 Brazil 0800 11 0575 Canada 800 263 6236 China 10 6849 2578 Denmark 80 30 17 40 France 0800 23 20 79 Germany 0800 083 0902 Hong Kong 2407 8450 India 11 577 3282 Italy 02 98 22 201 Japan 03 3663 7974 The Netherlands 0800 099 3310 New Zealand 0800 600 200 Norway 00800 5456 5456 Spain amp Portugal 900 181 461 Sweden 020 26 34 52 Switzerland 0800 848 800 Taiwan 2 2651 6156 UK 0800 838 380 For other countries see our website www invitrogen com
10. Cycles Ramp time Acquisition UDG reaction 50 2 min 1 No UDG inactivation template denaturation 95 2 min 1 No Denaturation 94 5 sec No Hybridization 55 10 sec 40 50 Single acquire Elongation 72 10 sec No Melting curve 95 0 sec 55 15 sec 95 0 sec 0 1 C sec Continuous 40 0 sec Uracil DNA glycosylase mediated carry over prevention Protocol 1 Program the LightCycler as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 18 ul of the master mix to each capillary Add 2 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the capillary 5 Gently mix and make sure that all components are at the bottom of the tube Centrifuge briefly 6 Place reactions in the thermal cycler programmed as described above Collect and analyze the results Continued on the next page Tips and Recommendations for Programming the Instrument Follow the instructions in the LightCycler user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument e The LightCycler can only be used with FAM label
11. PRISM is a registered trademark of Applied Biosystems LightCycler is a registered trademark of a member of the Roche Group iCycler is a trademark of Bio Rad Laboratories Rotor Gene 3000 is a trademark of Corbett Research Smart Cycler is a registered trademark of Cepheid Opticon is a registered trademark of MJ Research Inc Mx3000P is a trademark of Stratagene ABI PRISM 7700 TM The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the ABI PRISM 7700 See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 50 yl Monoplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 10uM 1u 50 ul 200 nM Unlabeled primer 10 uM 1ul 50 ul 200 nM ROX Reference Dye 50X 1u 50 ul 1X Autoclaved distilled water to 40 ul to 2000 pl Multiplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 1 10 uM 0 5 ul 25 ul 100 nM Unlabeled primer 1 10 uM 0 5 ul 25 ul 100 nM LUX labeled primer 2
12. R plate Add 10 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube strip plate 5 Gently mix and make sure that all components are at the bottom of the tube well Centrifuge briefly if needed 6 Place reactions in the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument Follow the instructions in the ABI PRISM 7700 user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument e Choose the Single Reporter plate format when opening a new plate to read e To set up the plate define all of the wells that contain reactions and then select the appropriate Dye Layer FAM or JOE for each Sample Standard or NTC e Note that as long as you select a well with at least one dye you can always change the dye layer parameter after analysis Unselected wells will not be read e You can manually choose the cycling Threshold setting in the Amplification Plot after a reading e By default the baseline Start Cycle is 6 and the End Cycle is 15 but you should change these to reflect the actual baseline of your specific amplification profile e To analyze the melting curve go to the File menu and select Export gt Multicomponent to create a multicomponent file Then open the multicomponent file usin
13. ce pipetting errors For each reaction add 40 ul of the master mix to each tube or qPCR plate well white tubes plates are preferred Add 10 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube plate 5 Gently mix and make sure that all components are at the bottom of the tube well Centrifuge briefly if needed 6 Place reactions in the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument Follow the instructions in the DNA Engine Opticon 2 user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument e Besure to select all wells in which there is a reaction The reaction type can be changed after acquisition e For monoplex reactions Select Single and then describe the plate 50 ul MJ white plate Dye 1 FAM and Dye 2 FAM e For multiplex reactions Select Both and then describe the plate 50 ul MJ white plate Dye 1 FAM and Dye 2 JOE e For Quantitation analysis Select Autoscale Smooth Subtract baseline and Average over cycle range cycle range 1 50 and select the threshold manually e For Melting Curve analysis Display dI dT and select Subtract baseline Global minimum and a temperature range of 60 60 Peak location boundaries should be 75 90 e The JOE reading is d
14. cript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions 11 Cepheid Smart Cycler TM The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the Cepheid Smart Cycler See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 20 yl Monoplex reaction Component Initial conc Single rxn 20 ul 50 rxns 20 ul Final conc Platinum qPCR SuperMix UDG 2X 125 pl 625 ul 1X LUX labeled primer 10uM 0 5 pl 25 ul 200 nM Unlabeled primer 10 uM 0 5 ul 25 ul 200 nM Autoclaved distilled water to 18 ul to 900 ul Cycling Program See the following page for tips and recommendations on programming the Smart Cycler P Acquisition Action Temp C Time Cycles UDG reaction 50 2 min 1 No UDG inactivation template denaturation 95 2 min 1 No Denaturation 95 15 sec No Hybridization 50 30 sec 40 50 Yes Elongation 72 30 sec No Melting curve analysis 60 95 Yes Uracil DNA glycosylase mediated carry over prevention Protocol 1 Program the Cepheid Smart Cycler as shown in the cycling program Optimal cycling temperatures and times may vary for different target
15. ed LUX Primers e FAM is acquired in the F1 channel 4 e Multiplex reactions are not available on the LightCycler Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCb 11730 025 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions Bio Rad iCycler The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the Bio Rad iCycler See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 50 yl Monoplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 10uM 1u 50 ul 200 nM Unlabeled primer 10uM 1ul 50 ul 200 nM Autoclaved distilled water to 40 ul to 2000 pl Cycling Program See the following page for tips and recommendations on programming the iCycler Recommended Action Temp C Time Cycles Ramp time Acquisition Cycle 1 UDG reaction 50 2 min 1 Yes Cycle 2 UDG inactivation template denaturation 95 2 min 1 Yes Cycle
16. es Denaturation 95 15 sec 40 50 Yes Hybridization Elongation 60 30 sec Yes Melting curve analysis click on Add Dissociation Stage button 95 60 95 Yes Uracil DNA glycosylase mediated carry over prevention Continued on the next page 18 Cycling Program continued Alternative Action Temp C Time Cycles Ramp time Acquisition UDG reaction 50 2 min 1 Yes UDG inactivation template denaturation 95 2 min 1 Yes Denaturation 95 15 sec Yes Hybridization 55 30 sec 40 50 Yes Elongation 72 30 sec Yes Melting curve analysis click on Add Dissociation Stage button 95 60 95 Yes Uracil DNA glycosylase mediated carry over prevention Protocol 1 Program the ABI PRISM 7900 as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets 2 Prepare a master mix of all components except template as specified Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 8 ul of the master mix to each tube strip well or well of the qPCR plate Add 2 ul of sample template 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA to each reaction vessel and cap or seal the tube strip plate 5 Gently mix and make sure that all components are at the bottom of the tube well Centrifuge briefly if needed 6 Place reactions i
17. g the Dissociation Curve software program from ABI Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCb 11730 025 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions Roche LightCycler TM The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the Roche LightCycler See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 20 yl Monoplex reaction Component Initial conc Single rxn 20 ul 34 rxns 20 ul Final conc Platinum qPCR SuperMix UDG 2X 10 ul 340 ul 1X LUX labeled primer 10 uM 1u 34 ul 500 nM Unlabeled primer 10 uM 1 ul 34 ul 500 nM Platinum Tag DNA Polymerase 5 U ul 0 12 ul 4ul 1 2 U BSA nonacetylated ultrapure 5 mg ml lu 34 ul 250 ng ul Autoclaved distilled water to 18 ul to 612 ul Final concentration includes Platinum Taq from Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the LightCycler Recommended Action Temp CC Time
18. ifuge briefly if needed 6 Place reactions in the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument Follow the instructions in the ABI PRISM 7000 user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument e Check the light bulb intensity of the instrument monthly as instructed in the user manual The intensity value should be gt 100 If the intensity is too low data acquisition will be adversely affected e When using both FAM and JOE labeled primers on the same plate but in different wells i e in monoplex you should select both FAM and JOE as the dye for all wells In general for monoplex reactions we recommend using just one dye per plate because the analysis will look better e Use the following filters for each dye type FAM Filter A JOE Filter B ROX Filter D e To improve the quality of the LUX signal you can increase the amount of ROX to 2 5 ul per 50 ul reaction e You can manually choose the cycling Threshold setting in the Amplification Plot after a reading e By default the baseline Start Cycle is 6 and the End Cycle is 15 but you should change these to reflect the actual baseline of your specific amplification profile e The default parameters for analyzing the melting curve are acceptable Additional Products Product Catalog number Size Plati
19. l 1 5 ul 3 mM Autoclaved distilled water to 40 ul to 20 ul 6 mM total Mg including MgCh in Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the Corbett Rotor Gene 3000 Recommended Action Temp CC Time Cycles Ramp time Acquisition UDG reaction 50 2 min 1 No UDG inactivation template denaturation 95 2 min 1 No Denaturation 95 5 sec 40 50 No Hybridization Elongation 60 10 sec Yes 1 C step default wait 60 sec on first step Melting curve analysis 60 95 and 5 sec step after Yes Alternative Action Temp C Time Cycles Ramp time Acquisition UDG reaction 50 2 min 1 No UDG inactivation template denaturation 95 2 min 1 No Denaturation 95 5 sec No Hybridization 55 10 sec 40 50 Yes Elongation 72 10 sec No 1 C step default wait 60 sec on first step Melting curve analysis 60 95 and 5 sec step after Yes Uracil DNA glycosylase mediated carry over prevention Continued on the next page 10 Protocol 1 5 6 TM Program the Corbett Rotor Gene 3000 as shown in the cycling program Optimal cycling temperatures and times may vary for different target sequences and primer sets Prepare a master mix of all components except template as specified The 50 pl final reaction volume is suitable for 36 position rotors and the 25 1 final reaction volume is suitab
20. late Gently mix and make sure that all components are at the bottom of the tube well Centrifuge briefly if needed Place reactions in the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument TM Follow the instructions in the Stratagene Mx3000P user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument For a good signal with JOE labeled primers change the acquisition gain for the JOE filter from 1 X to 4X or 8X The ROX Reference Dye should be diluted up to 1 10 prior to addition to the master mix to enable accurate pipetting For quantitative analysis select Adaptive Baseline Algorithm Amplified Base Threshold and 3pt Moving Average as the default parameters If there is too much background adjust the baseline manually Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCb 11730 025 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions 17 ABI PRISM 7900 The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the ABI PRISM 7900 See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional pr
21. le for 72 position rotors Note Preparation of a master mix is crucial in quantitative applications to reduce pipetting errors For each reaction add 40 ul 36 position rotor or 20 ul 72 position rotor of the master mix to each tube Add 10 ul 36 position rotor or 5 ul 72 position rotor of sample template to each reaction vessel and cap the tube The sample template may be 100 pg to 1 ug of genomic DNA or cDNA generated from 1 pg to 1 ug of total RNA Gently mix and make sure that all components are at the bottom of the tube Place reactions in the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument Follow the instructions in the Corbett Rotor Gene 3000 user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument Select the checkbox to automatically perform calibration for FAM or JOE dye before each run at 60 C For analysis select Linear Scale and then Autoscale The default parameter should be Dynamic Tube a Slope Correct Then select Autofind Threshold Optionally you can select Ignore First Cycle The default parameters are acceptable for analyzing the melting curve Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCl 11730 025 500 reactions SuperS
22. n the thermal cycler programmed as described above Collect and analyze the results Tips and Recommendations for Programming the Instrument Follow the instructions in the ABI PRISM 7900 user manual to program the instrument and analyze results The following are tips and recommendations for programming the instrument Choose the Absolute Quantification plate format when opening a new plate to read Also make sure to select the appropriate Container from the drop down menu To set up the plate click the Add Detector button and select the appropriate detectors for the plate e g FAM JOE any custom detector settings that you create Click the Copy to Plate Document button then click Done Select all of the wells that contain a particular marker and then check the Use box on the Setup menu You can then assign a Task for each well Sample Standard or NTC Click on the Instrument tab to make any changes to the Thermal Profile Click Add Dissociation Stage to do melt curve analysis of your reactions The default parameters are acceptable You can choose the cycling Threshold and Baseline settings in the Amplification Plot manually or automatically The individual reaction volume can be reduced to 5 ul if necessary e g if you are using 384 well plates Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCl 11730 02
23. num Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCl 11730 025 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions Corbett Rotor Gene 3000 TM The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the Corbett Rotor Gene 3000 See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page Note that the 50 ul final reaction volume is suitable for 36 position rotors and the 25 ul final reaction volume is suitable for 72 position rotors Monoplex reaction Component Initial conc Single rxn 50 ul Single rxn 25 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 12 5 ul 1X LUX labeled primer 10 uM 11d 0 5 ul 200 nM Unlabeled primer 10 uM 1u 0 5 pl 200 nM Autoclaved distilled water to 40 ul to 20 ul Multiplex reaction Component Initial conc Single rxn 50 ul Single rxn 25 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 125 ul 1X LUX labeled primer 1 10 uM 0 5 ul 0 25 ul 100 nM Unlabeled primer 1 10 uM 0 5 ul 0 25 ul 100 nM LUX labeled primer 2 10 uM Tul 0 5 ul 200 nM Unlabeled primer 2 10 uM 11d 0 5 ul 200 nM MgCb 50 mM 3 p
24. one in the TET channel You may need to recalibrate the machine to clean up the data The recalibration protocol is included in the Opticon 2 manual Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCl 11730 025 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reactions 11732 088 500 reactions 15 Stratagene Mx3000P The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the Mx3000P See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 50 yl Monoplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 10uM 1u 50 ul 200 nM Unlabeled primer 10uM 1ul 50 ul 200 nM ROX Reference Dye diluted 1 10 Dilute 1 10 1g 50 ul 1X Autoclaved distilled water to 40 ul to 2000 pl Multiplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 1 10 uM 0 5 ul 25 ul 100
25. ons 11732 088 500 reactions ABI PRISM 7000 The following real time quantitative PCR settings and protocol are designed for using LUX Primers on the ABI PRISM 7000 See the LUX Fluorogenic Primers manual available at www invitrogen com lux for more information and additional protocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 50 yl Monoplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 10uM 1j 50 ul 200 nM Unlabeled primer 10 uM 1u 50 ul 200 nM ROX Reference Dye 50X 11d 50 ul 1X Autoclaved distilled water to 40 ul to 2000 pl Multiplex reaction Component Initial conc Single rxn 50 ul 50 rxns 50 ul Final conc Platinum qPCR SuperMix UDG 2X 25 ul 1250 ul 1X LUX labeled primer 1 10 uM 0 5 ul 25 ul 100 nM Unlabeled primer 1 10 uM 0 5 ul 25 ul 100 nM LUX labeled primer 2 10 uM 0 5 pl 25 ul 200 nM Unlabeled primer 2 10 uM 0 5 ul 25 ul 200 nM MgCl 50 mM 3 pl 150 ul 3 mM ROX Reference Dye 50X 1 ul 50 ul 1X Autoclaved distilled water to 40 ul to 2000 ul 6 mM total Mg including MgCb in Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the ABI PRISM 7700 Recommended
26. otocols Master Mix Prepare a Master Mix as described below then add template to each reaction as described in the protocol on the following page The standard reaction volume is 10 yl Monoplex reaction Component Initial conc Single rxn 10 ul 50 rxns 10 ul Final conc Platinum qPCR SuperMix UDG 2X 5 pl 250 ul 1X LUX labeled primer 10uM 0 2 ul 10 pl 200 nM Unlabeled primer 10 uM 0 2 ul 10 pl 200 nM ROX Reference Dye 50X 0 2 ul 10 ul 1X Autoclaved distilled water to 8 ul to 400 ul Multiplex reaction Component Initial conc Single rxn 10 ul 50 rxns 10 ul Final conc Platinum qPCR SuperMix UDG 2X 5 pl 250 ul 1X LUX labeled primer 1 10 uM 0 1 ul 5 ul 100 nM Unlabeled primer 1 10 uM 0 1 ul 5 ul 100 nM LUX labeled primer 2 10 uM 0 2 ul 10 ul 200 nM Unlabeled primer 2 10 uM 0 2 ul 10 ul 200 nM MgCl 50 mM 0 6 ul 30 ul 3 mM ROX Reference Dye 50X 0 2 ul 10 ul 1X Platinum Tag DNA Polymerase 5U 0 12 ul 6 pl 0 6 U Autoclaved distilled water to 8 ul to 400 ul 6 mM total Mg including MgCh in Platinum qPCR SuperMix UDG 1 2 U total Platinum Taq DNA Polymerase including 0 6 U in Platinum qPCR SuperMix UDG Cycling Program See the following page for tips and recommendations on programming the ABI PRISM 7900 Recommended Cycling Program Action Temp C Time Cycles Ramp time Acquisition UDG reaction 50 2 min 1 Yes UDG inactivation template denaturation 95 2 min 1 Y
27. r source Do not use the external well factor plate for calibration Make sure that the first two cycles are programmed as separate cycles and not as two steps within a single cycle The iCycler will automatically calibrate at or above 90 C immediately preceding the first cycle containing a temperature above 90 C and this will kill the uracil DNA glycosylase The iCycler must be calibrated for the specific dye used FAM or JOE Even though the software will list all the dyes while setting up a plate this does not mean that the instrument has been calibrated for these dyes To view the dyes for which the instrument is calibrated go to the C Program Files Bio Rad iCycler Ini folder and open the RME ini file in a text editor such as Notepad In the file you should see for example FAM 490 490 20X_ _530 30M 4 021013e 003 This indicates that the instrument has been calibrated for FAM Refer to the iCycler manual or call Bio Rad technical service for help with calibrating the dyes For monoplex reactions we recommend using just one dye type per plate as the data will look better for analysis Multiplex reactions are currently not recommended on the iCycler Additional Products Product Catalog number Size Platinum Quantitative PCR SuperMix UDG 11730 017 100 reactions includes separate tubes of ROX Reference Dye and MgCl 11730 025 500 reactions SuperScript III Platinum One Step qRT PCR Kit 11732 020 100 reacti
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