Hello, I want to use a multiple(2) fragments In-fusion cloning technique, so I tried to design four primers with snapgene and takara online tool, but all of the primers have a Tm more than 65 °C (one of them is 72°C) no matter how I alter the primers the result wont change, what can I do about it? is it going to cause a problem?
Hey Sepideh. 72 is ok to be the maximum Tm for your primers. But having more than 5 degrees as a difference between your primers Tm is a problem. try to find new areas on your genome to make more uniform primers Tm. or you can try reducing your primers length( not less than 18 bp) or increasing it ( not more than 28 bp) to have new Tm. If you can NOT achieve this, you can order your primers and use two different annealing temperatures on the thermocycler machine for each set of primers.
I'd just give it a try. Primers are cheap. Unless you are trying to detect tiny amounts of target DNA in a complex mixture like an environmental or clinical sample, you can get away with quite a lot of deviation from the ideal.
If there is a big difference between the primer Tm's it might be a good idea to try lowering the concentration of the more stable primer. In any case, doing a primer titration using all combinations of 50, 150, 300, 600 nM of each primer is worth doing.
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