Second Strand cDNA Synthesis Kit, 200 reactions - FAQs

查看更多产品信息 Second Strand cDNA Synthesis Kit - FAQs (A48571, A48570)

4 个常见问题解答

What is the correct reaction temperature that I should use for second strand cDNA synthesis using the Second Strand cDNA Synthesis Kit?

The second strand reaction must be incubated at 16 degrees C to prevent spurious synthesis by DNA polymerase I. At higher temperatures, DNA polymerase I tends to strand-displace rather than nick translate.

When using the Second Strand cDNA Synthesis Kit, what steps do I need to take to properly prepare the second strand reaction mixture?

Dilute the first-strand reaction as outlined in the manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019413_SecondStrandcDNASynthesisKit_UG.pdf) on page 2 under Second Strand cDNA Synthesis step. Failure to properly dilute the first-strand reaction changes the pH of the second strand reaction and influences the activity of the 3'-5' and 5'-3' exonuclease activities of DNA polymerase I.

When using the Second Strand cDNA Synthesis Kit, how can I avoid incomplete RT inactivation?

Always perform the RT inactivation step after first-strand cDNA synthesis.

I am getting low yield of second strand cDNA product using the Second Strand cDNA Synthesis Kit. What is the reason?

The main reasons are incomplete RT inactivation, improper preparation of the second strand reaction mixture, and incorrect reaction temperature.