I have to perform a PCR reaction with degenerate inosine containing primers, for virus typing purposes. I have the primers and protocol aviable for degenerate conditions. But in the manuel of PCR kit which I intend to use, it is written - "do the anealing step at 60 degreaces". And the question is - can I use this kit, lowering the annealing temperature according degenerate protocol conditions, or I have to search another PCR kit for this reaction?
Hi Irina
Usually a PCR kit manufacturer may recommend some temperatures but you may change it based on your needs to some extent. Can you please specify your kit?
Thank you
Thank you, Ali.
The kit is AgPath-ID™ One-Step RT-PCR (Applied Biosystems™).
Hi,
there should be several kits which get along with this degenerated primers. I have optimised some assays with Quantinova Pathogen an this works quite well. Depending on the annealing temperatue you might need to use a 3-Step cycling but normally 2-Step should work just fine
Good luck!
Sven
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