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problem in getting results from a optimized PCR - (May/25/2007 )

hi everyone
i have optimized a PCR for a gene it gave really good results with different samples but now i m facing a big problem that when i used do the PCR reaction with the same conditions i get the smear instead of a proper band of DNA, i have already changed my Primer dilution twice, buffer, Taq polymerase enzyme, dNTPs, water but i m getting the same results even i have tried to re-amplify the PCR which i get positive there there was a clear band but again there is only smear.
i m very worried about it because i have to submit a report to my supervisor as soon as possible, can any one help me in this regard

-sabahat Khaliq-

How about your initial DNA template?


Get new aliquots of all solutions and retry the PCR. Check template by running on gel to make sure its not degrading.


Hi, If the situation is really as what you described here, I think the problem is of your template.


I agree with the others about template, but I suggest you also to check the conditions of your electrophoresis run.
How is your ladder run? Have you always kept on using the same solutions (e.g. loading buffers, TBE/TAE, sybr green etc)
Hope this could help!



for me it sounds like contamination. what about the negative control? do you also have a kind of positive control. it also coud be degradation. but it will be possible to resolve this two problems. is it possible for you to get another DNA (new isolation) or is your template limited?

good luck