Hello
I am trying to exchange the polymerase in a PCR method for genotyoping of a gene, to Qiagens HotStarTaq Plus DNA Polymerase.
My problem is that my homozygote samples, that are either wildtype or knockout, have nice clear and visible bands. However for the heterozygote samples, the wildtype band (WT) is much weaker and much less visible than the knockout band, as seen on the picture below. Except for sample 26255 the one next to the marker, a heterozygote sample that has a strong and visible wildtype.
Do you guys have any idea as to why that one heterozygote sample has a very clear wildtype band, when the other heterozygote samples don't?
And do you have any suggestions for how I could optimize on this?
It is not possible for us the stabiliza the DNA concentration across the samples, and it is also not possible for us to meassure how high the DNA concentration is in the samples.
Hope to hear from you.
He meant by "Ta" annealing temperature.
I do not think that the difference in amplification efficiency between WT and KO bands comes from the size of the amplicons, they are too close.
It looks like the KO band is easier to amplify compared to the WT band: the polymerase may have some difficulties to amplify the WT. Did you check the GC content of the WT? is it high i.e. over 60%. Would it be possible that the WT forms strong secondary structures at the annealing temperature? Such type of things may account for the less efficient amplification of the WT band relative to the KO band.
I would suggest you to add DMSO to your PCR at a 5% final concentration.
This will help amplifying your WT fragment in case of high GC content or secondary structures. I also would recommend to increase the denaturation time, like 3 to 4 min at first and then 1 min for each cycle.
I hope it helps
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