I have tried doing a PCR with kapa as well as Phusion HF polymerase. I am getting amplification with kapa, but Phusion is just not working. Both the master mixes use same: 1.5mM MgCl2 and 0.2mM dNTPs. With Phusion, we were primer:DNA is only variation. Please suggest the ideal ratio for primer to template..
Hi,
The exact ratio of primer to DNA should not decide about success or failure of your reaction. According to the supplier's protocol:
https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530
you need a final primer concentration of about 0.5 µM per primer and less than 250ng of template in your reaction. The amount of template could be an issue, but the ratio of primer to template DNA should not be that important. However, there should be an optimal ratio of both components, but your PCR probably failed due to other reasons. Are the temperature and time settings in your PCR program in accordance with the supplier's protocol?
Good luck!
Ok! Yes, we are setting gradient annealing according to NEB protocol.
Thanks for your reply
The HF polymerases can be really picky about temperature, annealing time and a whole bunch of other stuff. I find that adding a little DMSO to the PCR reaction helps a lot with HF polymerases and also testing your annealing temp and time can help a lot.
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