Real-Time PCR Set-up Instructions (for abm Reverse Transcription Products)

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1 Real-Time PCR Set-up Instructions (for abm Reverse Transcription Products) The following protocol is for real-time PCR using abm s EvaGreen qpcr MasterMix (Cat. No. MasterMix-R, -LR, -ic and -S). Please refer to the EvaGreen qpcr MasterMix Selection Guide Version.0 ( EvaGreen qpcr MasterMix Selection Guide-Web.pdf) for selecting the appropriate qpcr MasterMix formulation applicable to your instrument model. The reverse transcription reaction prior to qpcr can be prepared from using any of the abm s EasyScript TM and EasyScript Plus TM Reverse Transcriptase Products listed below. Cat. No. Product Name G23, G232 EasyScript TM Reverse Transcriptase G77, G237 EasyScript TM Plus Reverse Transcriptase G233, G234, G49 EasyScript TM Kit G235, G236 EasyScript TM Plus Kit G45, G452 EasyScript TM SuperMix G453, G454 EasyScript TM Plus SuperMix G485, G486, G490, G492 5X All-in-One MasterMix abm Reverse Transcription Selection Guide Kit Flexible priming using abm s EasyScript and EasyScript Plus Kit EasyScript or EasyScript Plus Oligo (dt) / Random Primers / Gene Specific Primers dntps RNaseOFF Ribonuclease Inhibitor 5X buffer abm G233, G234, G49, G235, G236 SuperMix Minimize pipetting errors with abm s EasyScript and EasyScript Plus SuperMix EasyScript or EasyScript Plus 2X Reaction Mix abm G45, G452, G453, G454 All-in-One MasterMix Ultimate convenience and reproducibility using abm s 5X All-in-One MasterMix 5X All-in-One MasterMix abm G485, G486, G490, G492

2 <Real-time Set-up using ABI StepOne System> * Recommended EvaGreen 2X qpcr MasterMix for ABI StepOne is MasterMix-R. 3. Start the qpcr Reaction. The Quantitation-Comparative CT (ΔΔCT) standard protocol as shown below is recommended. Optimize the reaction condition for primers with Tm higher or lower than 60 C. Enzyme Activation Denaturation, Annealing and Extension 4. Check the amplification and melting curves after the reaction is completed. When using ABI StepOne System, please refer to its instruction manual for the analytical methods.

3 <Real-time Set-up using Roche LightCycler480 System> * Recommended EvaGreen 2X qpcr MasterMix for Roche LightCycler480 is MasterMix-S. 3. Start the qpcr Reaction. The standard protocol as shown below is recommended. Optimize the reaction condition for primers with Tm higher or lower than 60 C. 4. Check the amplification and melting curves after the reaction is completed. When using Roche LightCycler480 System, please refer to its instruction manual for the analytical methods.

4 <Real-time Set-up using BioRad iq5 Sytem> * Recommended EvaGreen 2X qpcr MasterMix for BioRad iq5 is MasterMix-iC. 3. Start the qpcr Reaction. The cycle+melt standard protocol as shown below is recommended. Optimize the reaction condition for primers with Tm higher or lower than 60 C. 4. Check the amplification and melting curves after the reaction is completed. When using BioRad iq5 System, please refer to its instruction manual for the analytical methods.

5 <Real-time Set-Up using ABI 7300 Sytem> * Recommended EvaGreen 2X qpcr MasterMix for ABI 7300 is MasterMix-R. 3. Start the qpcr Reaction. The standard protocol as shown below is recommended. Optimize the reaction condition for primers with Tm higher or lower than 60 C. 95 C 60 secs 4. Check the amplification and melting curves after the reaction is completed. When using ABI 7300 System, please refer to its instruction manual for the analytical methods.

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