Dear all,
I am amplifying a target sequence of 65 bases, which is a cDNA sequence now. For some reasons, my PCR primer can only bind to the target sequence with 11 bases in the first PCR cycle. Can anyone please tell me how I should set the annealing temperature for the first cycle? My enzyme is Hot start Taq DNA polymerase from NEB(M0495S). The optimal amplification temperature is: 95C denaturation, 45-68C annealing, 68C extension.
Thanks
Binbin Cui
Hi, your primer is quite short and the target sequence is very short as well. I would suggest you to run PCR optimization first to find out the best PCR condition for your case. To do the optimization, you may have different reactions set at different annealing temperature and duration.
For example:
Reaction 1 annealing temperature is set at 50 degree celsius for 30 seconds.
Reaction 2 annealing temperature being set at 55 degree celsius for 30 seconds.
Reaction 3 annealing temperature is set at 50 degree celsius for 1 minute.
and etc...
And then u check the PCR quality using conventional gel electrophoresis and the reaction which gives you the most intense, single band would be the best PCR condition..
Hi, what are these reasons, do they prevent you from simply making a longer primer? You can try very low annealing-temp, like 40-45 degrees, but most likely it won't work. In case you just want to clone this, you could directly order it as oligos that you anneal and clone directly, since you say it is only 65 nucleotides
fortunately cDNA is a much smaller genome than genomic dna so a low first cycle annealing temperature will still amplify quite cleanly. For a sequence of 11 bases or less the simple rule 4 xnumber of c and g plus 2c x Number of A and T works well but really the best idea is to run a gradient of annealing temperatures around the value you get from the 4xC+G + 2xA+T formula then after the first few cycles you can increase the annealing temperature to that of the full length primer if it is longer than 11 bases
Dear Binbin Cui,
In my eyes it is not possible to make a well working PCR with this very short primers. You would get a lot of unspecific amplification products! - For what reason you can not use longer primers?
Such short primers, you can use as sequencing primers! :-)
Best regards,
Thomas
Dear all,
My PCR primer is 19 bases long. But in the first cycle, only the last 11 bases of my primer can bind to the 3'-end of the cDNA molecule, which is around 60 bases long.
Best regards
Binbin Cui
Dear Binbin Cui,
A PCR with 19mer primers will work!
11 matching bases on the 3'-end will be enough!
Good luck!
Best regards,
Thomas
Thank you all for answering the question！
It works now after I set the annealing temperature of first cycle as 37C.
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