Hello everyone,
I did several colony PCR, the enzyme I used here was Taq. In my first colony PCR, I set annealing temperature as 65 degree (which was similar to my basic PCR using KAPA, the primers in both basic and colony PCR were the same), and I got no band for my target gene. Then in my second colony PCR, I modified annealing temp to 55 degree, and I got my gene band.
What I do not understand here is that annealing temp is for primer binding, in case that I used the same gene, the same primers, only change the enzyme, why I need to change annealing temp? what is the relationship between annealing temperature and enzymes?
Sorry if this question is silly. Thank you very much!
"why I need to change annealing temp"
>> you don't. Since your temperature changes does not show any logic and rather seems like abrupt guessing, the PCR failure could have been caused by something else.
>> Further, you are generalizing based on just single PCR and in single conditions, which should not be done.
>> Although, polymerases have different optimum temperatures and different efficiency, it does not cause complete failure of PCR reaction.
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