Hello,
I have been having trouble with smearing PCR product of a type of marker in my gel lately (picture is attached). I'm working with a triploid organism, so three bands can be observed, but the smearing sometimes make it difficult to see bands. The samples in each well are the same, but used in different temperature from 57-64 degree Celsius from left to right. This is just to show that my smearing problem is not due to inappropriate annealing temperature. The amount of DNA loaded in each well is 10ul in a 0.5 cm wide well. Anybody has any suggestion for this problem?
Dear Minh,
I understand from your question that you expect to see only 3 bands, right? From the gel, I can see you have more than the expected number of bands and not just a smear. What I normally do when I encounter such is to:
1. Change the Taq or
2. Add betaine (1M in final concentration) or DMSO (1%).
Hope this helps.
Regards,
Israel
Dear Minh, try to decrease Mg concentration first. The advices of Israel are useful too. However I recommend You to do all optimization in combination with Ta gradient.
It may be due to non-specific amplification. To do away with these, the most probable solution will be to increase the annealing temperature in order to eliminate the non-specific amplicons.
Elvis Ndzi Thank you for your response. The picture I have shown is a result of gradient PCR. The highest temperature goes to 64 degrees, and yet these non-specific band still show up. I don't think that the optimal annealing temperature would exceed 64
Dear Minh T. Le, sorry for my response, I never read through your question to the end, I could have seen that you already mentioned you did a gradient PCR with varying temperature. You could try reducing the concentration of Mg2+ or changing the enzyme aliquote you used.
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