I am in the process of preparing to do a RACE-PCR experiment and am testing some primers for this. I have designed one forward primer and two reverse primers (these will be used in a nested approach in the RACE-PCR). When I ran the PCR reaction today to check fragment size amplification on plasmid DNA containing the gene of interest, one forward/reverse mix gave a strong band as expected (~130bp), whereas the other mix did not give the expected band (at ~180bp).
These primers look good on Primer Blast (good specificity to the target, low self complementarity etc.) with similar Tms of ~64C.
The conditions I used for the experiment were as follows:
95C for 2 min
25 cycles x 95C for 30s, 60C for 30s, 72C for 30s
72C for 2 min
I am still confused why I am getting such strong amplification with one primer set and no band whatsoever with the primer stock. Could anyone give any suggestion to improving my cycling protocol or any other points to consider? Something that may or may not be relevant is that I do get a fairly strong primer-dimer band at the bottom of each lane, I am probably including too much primer into the mix, but this is something that is consistent between the successful and unsuccessful reaction.
Hi Rachel,
The theoretical calculations of primer properties are 'still theoretical' ...I'll sugegst you to do a gradient PCR for the other set of primers separately. In the reaction the optimum primer may slightly vary..try it.
bye
Hi Rachel,
Just to check a few things about your primers: you say that your primers have low self-complementarity, but did you check for trans-complementarity with each other, i.e. possible formation of primer dimers? If not, this is worth doing. It may also be worth checking the structure of the template in case there's a particularly strongly-structured region or some other difficult-to-copy sequence (e.g. G-rich) sequence between the sites of your two reverse primers that may be causing problems. Is your sequence particularly biased towards one or more bases?
Ajay's idea of a gradient PCR is a good one, but there's also a few other possible things you can try. One other solution to improve your PCR might be to add a small amount of DMSO to the reaction mixture, which can help replicate through more highly-structured or complementary sequences, or support partially complementary primers. Otherwise, it might just be quicker to order a new reverse primer I'm afraid!
Good luck!
Dan
Hi all, I have tried adding DMSO with no luck, and will try the gradient PCR today.. if no success there I will design a new primer. Thanks for your help!
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