Team:Calgary/Notebook/Protocols/Process3
From 2011.igem.org
(Difference between revisions)
Emily Hicks (Talk | contribs) |
|||
Line 4: | Line 4: | ||
BODY=<html> | BODY=<html> | ||
+ | <p>The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, is used to generate cDNA from isolate RNA (from <i>Pseudomonas spp.</i>) using reverse transcriptase.</p> | ||
<ol> | <ol> | ||
<li>Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li> | <li>Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li> |
Revision as of 02:58, 29 September 2011
cDNA Synthesis/Reverse Transcription Reaction
The following protocol is found in the Quanta qScript Reverse Transcription Kit manual, is used to generate cDNA from isolate RNA (from Pseudomonas spp.) using reverse transcriptase.
- 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:
- Mix components by gently vortexing and then centrifuge 4s to collect contents.
- Incubate in a PCR machine using the following conditions:
- 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).
Component | Volume |
RNA (40ng – 10ng to 1μg total RNA) | variable |
Nuclease free water | variable |
qScript cDNA synthesis mix | 4 μL |
Final Volume | 20 μL |