i am working on a SNP present on BCL11A by arms PCR on Thalassaemia Major Samples . Results were good then all of sudden no specific bands starts to appear. I have not change anything . And the problem is with major allele only . These non specific bands forming ladder like pattern. What's happening to my reaction kindly help. Gel pic is attached .
Hi Zeeshan,
very strange indeed. it's not clear on your picture and to be sure, do you run your ARMS by two multiplex PCR, one PCR with the wild type allele and the second for mutated, both with an internal PCR control? do those noisy bands reveal a small deletion associated with the major allele? could you compare wild type, heterosexual and homozygote just to see is the noise appears strictly with major? if it's just misPCR, you could increase your Tm by 2-3°c just to see if it disappear. in case it does not, I would sequence some samples just to be sure this pattern is not in linkage desiquilibrium with the major allele.
fred
if it's just starting, will you consider degradation of your primers, of your samples? or contamination?
in these cases I would restart from stock solutions and test quality of samples.
fred
Could be a contamination of one of the reagents. Try to do again with completely new reagents and new H2O.
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