Description
| Target |
cDNA Synthesis Kit |
| Tested Applications |
PCR |
| Storage |
Store at -20 °C for up to 18 months. Avoid repeated freeze/thaw cycles. |
| Buffer |
Enzyme: 20 mM Tris-HCl, pH 7.4, 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, 0.01% NP-40 and 50% glycerol. |
| Concentration |
200 U/µl |
| Directions for use |
Assay Procedure:
- Mix and heat the RNA Template, 2X Reaction Mix, and Primers to 65 °C for 5 minutes. Allow the RNA Template to stand in an ice bath for at least 1 minute.
- Add the following components to a PCR tube on ice:
| Component |
Volume |
| RNA Template |
Variable
10 pg – 2 µg total RNA or 10 pg – 500 ng mRNA) |
| 2X Reaction Mix |
10 µl |
| Primers |
1 µl |
| Enzyme Mix |
2 µl (200 U) |
| RNase Inhibitor |
1 µl (20-40 U) |
| Water |
Variable, up to 20 µl |
| Total Volume |
20 µl |
- Mix by gently pipetting up and down.
- Close the lid on the tube and incubate in a temperature-controlled water bath at 55 °C for 50 minutes for the extension step.
Note: the optimal temperature for extension is likely between 42-60 °C.
- Incubate the tube at 70 °C for 15 minutes to inactivate the Reverse Transcriptase before amplification.
Notes:
- Avoid cross-contaminating the RNA template (total RNA, synthetic RNA transcript, poly(A) + mRNA) with DNA.
- Recommended primers (per 20 µl reaction):
- 2.5 µM of oligo(dT) – anneal to 3′-poly(A) + mRNA
- 2.5 ng/µl of random primers – anneal at non-specific sites of RNA templates
- 2 µM of gene-specific primers
|
| Availability |
Shipped within 10-15 working days. |
| Note |
This product is for research use only. |
Research Articles on cDNA Synthesis Kit
