Hello all,
I am trying overlapping mutation for a site specific point mutation ( I tried with QuickChange kit, not working) . For which I have to first generate two fragments of my target gene. target gene is 603 bp and the fragment A is suppose to be 360bp. For the pcr for fragment A I use forward gene specific primer ( 30 bp length , tm - 69 degree) and reverse mutagenic primer ( 46 bp length, tm- 78 degree) but I did not get any band on the gel with gradient per from range 50-78 . I kept one positive control with only the gene specific primers so I got band exactly at 603 bp so I know my reaction setup is not the problem and every time I am getting primer dimer issue at the bottom.
64 degree is the calculated annealing temp. even In dat temp I am not getting any bands.
So, I tried touch down with from 70-62 degree 16 cycles 0.5 degree decrease each cycle and remaining 18 cycles with 64 degree so I started getting one band which is between 100 and 200 bp again not with the expected size. I don't know whether its an actual band or primer dimer.
Initially also I have faced this problem while amplifying a 400 bp target gene. but I have successfully amplified genes above 600.
Is it a normal problem with smaller fragments?
Your help will be much appreciated.
Thank you.
Image attached of the touchdown PCR gel
The primer is very long so may have secondary structure problems so you could try running a dmso gradient with 1% to 8% dmso (final concentration) and ideally with a hot start polymerase to minimise secondary structure in the primers
It looks like there is a lot of template showing up at the higher sizes, maybe try using less template material? I agree that DMSO is a good additive to try, betaine can help too. What size reaction volume are you running? Sometimes a smaller volume (20-25) can give a better yield than a larger volume (30-50) as you get faster temp changes in the tube. Your fragment size isn't that small so it should (in theory) amplify well.
Thank you Amy Klocko Paul Rutland for your recommendation. I will surely use DMSO in my next setup.
Paul Rutland Reaction volume is 25 ul.
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