cDNA Synthesis Kit
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(Size: 50 rxns)
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cDNA Synthesis Kit for optimal cDNA synthesis from a variety of RNA samples over a wide temperature range. The kit contains a M-MLV reverse transcriptase with extended thermostability and half-life and contains proprietary mutations for reduced RNase H activity. Kit Components:
Materials Required But Not Provided:
- Enzyme Mix: 100 µl
- 2X Reaction Mix: 500 µl
- Oligo dT20 Primer (50 µM): 50 µl
- Random Hexamer Primer (50 ng/µl): 50 µl
- Nuclease-Free Water: 1 ml
- Vortex Mixer
- Pipettes and Pipette Tips
- PCR Tubes
- Ice Water Bath
- Temperature-Controlled Water Bath (or equivalent)
Research Articles on cDNA Synthesis Kit
||cDNA Synthesis Kit
||Store at -20 °C for up to 18 months. Avoid repeated freeze/thaw cycles.
||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.
|Directions for use
- 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|
|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.
- 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
||Shipped within 5-12 working days.
||This product is for research use only.