I used all the reactions in the one tubes and share it in equal volume into separate tubes as the size of the sample . lastly template (cDNA) was added into all PCR tubes except in both positive and negative controls but after the PCR, all samples were developed bands which is not correct. I did the experiment 2 times to know the contamination source. I also did another experiment with the same template but primers are different and the result was good. I understand little bit where is the problem that is the primer maybe contaminated. is there such probability of primer contamination with cDNA?
Yes absolutely, given that your other primers showed desired PCR product. If you have repeated the experiments with new water, buffer, etc but still see unspecific band in your negative control (i.e. no DNA template) than your primers are contaminated.
In all likelihood you have amplicon contamination from previous experiments
Hi. Whenever you see bands for your negative control, it generally means there was some form of contamination. It probably means there is some template contamination. Any one of the reagents could be contaminated, or it could be the pipettor or tips.
The simple solution would be to re-setup the reaction using new reagents. If you want to find the source of the contamination, then you would need to change only one reagent at a time and set up a series of identical reactions where one reagent was change in each one. Filter tips could eliminate the possibility that the pipetter is the source of contamination. But you may also get by by cleaning the pipettor well since filter tips are more expensive.
Good luck.
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