From 2011.igem.org
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Revision as of 20:25, 27 September 2011
{{Team:Calgary/Notebookbar|
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Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.
Add the following to a 0.2mL thin-walled PCR tube sitting on ice:
TITLE=cDNA Synthesis/Reverse Transcription Reaction|
cDNA Synthesis by Reverse Transcription |
| Component |
Volume |
| RNA (40ng – 10ng to 1μg total RNA/rxn) |
variable |
| Nuclease free water |
variable |
| qScript cDNA synthesis mix |
4 uL |
| Final Volume |
20 uL |
Thaw all components (except enzyme), mix thoroughly and centrifuge before use.
Mix components by gently vortexing and then centrifuge 4s to collect contents.
Incubate in a PCR machine using the following conditions:
| 5 min at 25°C |
| 30 min at 42°C |
| 5 min at 85°C |
| Hold at 4°C |
After completion of cDNA synthesis, us 1/20th of the first-strand reaction (2-4μL) for PCR amplification in water. Also do 2,3, and 4-fold dilutions of each sample to ensure that the PCRs will be within the primer linear range. Do 3 replicates of each PCR reaction. Run PCRs on a real-time PCR machine (e.g. ABI 7900).
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