Team:Calgary/Notebook/Protocols/Process3
From 2011.igem.org
(Difference between revisions)
Emily Hicks (Talk | contribs) |
Emily Hicks (Talk | contribs) |
||
Line 4: | Line 4: | ||
BODY=<html> | BODY=<html> | ||
- | <li | + | <li>Thaw all components (except the enzyme/buffer mix), mix thoroughly and centrifuge before use.</li> |
- | <li | + | <li>Add the following to a 0.2mL thin-walled PCR tube sitting on ice:</li> |
<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
Line 36: | Line 36: | ||
</table> | </table> | ||
- | <li | + | <li value="3">Thaw all components (except enzyme), mix thoroughly and centrifuge before use.</li> |
- | <li | + | <li>Mix components by gently vortexing and then centrifuge 4s to collect contents.</li> |
- | <li | + | <li>Incubate in a PCR machine using the following conditions:</li> |
<div style="margin-bottom:60px"> | <div style="margin-bottom:60px"> | ||
Line 60: | Line 60: | ||
</tr> | </tr> | ||
</table> | </table> | ||
- | <li | + | <li value="6">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).</li></html>}} |
Revision as of 21:36, 27 September 2011
cDNA Synthesis/Reverse Transcription Reaction
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 |