Team:Calgary/Notebook/Protocols/Process3

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Revision as of 20:27, 27 September 2011

{{Team:Calgary/Notebookbar| TITLE=cDNA Synthesis/Reverse Transcription Reaction| BODY=

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:

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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 <li type="1">Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li>
 <li type="1">Add the following to a 0.2mL thin-walled PCR tube sitting on ice:</li>
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-<li type="1">Thaw all components (except enzyme), mix thoroughly and centrifuge before use.</li>
+<li type="1" value="3">Thaw all components (except enzyme), mix thoroughly and centrifuge before use.</li>
 <li type="1">Mix components by gently vortexing and then centrifuge 4s to collect contents.</li>
 <li type="1">Incubate in a PCR machine using the following conditions:</li>