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| Protocols for Realtime PCR |
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Reverse Transcription GPCL uses the High Capacity cDNA Reverse Transcription kit from ABI according to manufacturer’s instructions. Our standard methods are to use 800 ng total RNA in an RT reaction and to perform both a standard RT with enzyme and a negative control without enzyme. Other methods are available for quantitative reverse transcription. Superscript III (Invitrogen) iScript (BioRad) eAMV(RT) (Sigma) Transcriptor (Roche) Realtime PCR ABI Universal or Gene Expression Master Mix, for Taqman or SYBR green as appropriate, is used at final 1X concentration in a 25µl reaction volume on a 96 well plate. Assays on Demand are also used at a final 1X concentration as defined by the manufacturer. Custom designed primers are added at 250 nM final concentration and probes at 100 nM. GPCL standard procedure uses 20 ng cDNA equivalents per PCR reaction. GPCL routinely sets up 3 wells of PCR for each gene using each RT positive reaction and a single well for each gene using each RT negative control reaction. Cycling conditions: Standard cycling conditions for realtime PCR are: 95°C for 12 minutes 40 cycles of: 95°C for 15 seconds 60°C for 1 minute Please note clearly on the drop off form if other cycling conditions are required. |