Recently, I have done a PCR of a region of human p53 gene and got faint band after that. Then I used that PCR product as input and did a nested PCR with same PCR condition, but now the specific band disappeared and only smear was there. Can anybody help me to solve the problem. Why that happened and what should I do to get specific band.
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$\begingroup$ Can you tell us what the template DNA was in the first PCR? $\endgroup$– Alan BoydMay 30, 2017 at 11:18
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$\begingroup$ More detail of both protocols would be helpful. $\endgroup$– arboviralJun 29, 2017 at 11:21
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1 Answer
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Ther could be different problems affecting your r salts. Maybe the primers you are using are not specific enough or your PCR condition are not optimal. I would first check the primers by blasting them against the human genome to spot mismatches. If your primers do not bind uniquely to your gene of interest then design a new pair. If the primers are ok, then I suggest you to run a grandient PCR (annealing temperature from 50 to 65 degree Celsius) to determine the optimal annealing temperature of your primers. These two steps usually solve 99% of my PCR problems, assuming that your template DNA is ok and at the right concentration. If even with the gradient PCR you can't find a good condition, the next step is to play with the buffer by changing magnesium chloride concentration and maybe adding betaine.
