Protocol for First-strand cDNA Synthesis
(PCR template generation with M-MuLV Reverse Transcriptase)
The following protocol is optimized to generate first-strand cDNA for use in two-step RT-PCR.
Mix and briefly centrifuge all components after thawing, keep on ice.
- Add into sterile, nuclease-free tube on ice in the indicated order:
|Template RNA ||total RNA |
or poly(A) RNA
or specific RNA
|100 ng - 5 µg |
10 - 500 ng
0.01 pg - 0.5 µg
|Primer ||oligo(dT)18 |
or random hexamer
|0.5 µg (100 pmol) |
0.2 µg (100 pmol)
| ||DEPC-treated Water ||to 12.5 µl |
- Optional. If RNA template is GC rich or is known to contain secondary structures, mix briefly, centrifuge briefly and incubate at 65°C for 5 minutes, chill on ice, briefly centrifuge and place on ice.
- Add the following components in the indicated order:
Mix gently and centrifuge briefly.
- If oligo(dT)18 primer or gene-specific primer is used, incubate 60 minutes at 37°C.
If random hexamer primer is used, incubate 10 minutes at 25°C followed by 60 minutes at 37°C.
For transcription of GC rich RNA reaction temperature can be increased to 45°C
- Terminate the reaction by heating at 70°C for 10 minutes. Do not heat-inactivate enzyme prior to analysis of long cDNA to avoid cleavage.
- The reverse transcription reaction product can be directly used in PCR or stored at -20°C.
- Use 2 µl of the reaction mix to perform PCR in 50 µl volume.
- Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
Updated kovo 18, 2008 11:05