Hi,
During an experiment I have conducted series of Nested PCR and getting amplification in negative control but not in sample. Every troubleshooting measures have been taken. Also, Positive control which is plasmid, in that multiple bands were observed. What are those bands and why there is amplification coming in negative but not in samples? Multiple experiment repeats were done and every suitable troubleshooting methods have been applied (gel image attached last is positive and second last is negative control).
Can I please get the suggestions?
this result may caused by contamination of negative control while the gene in your samples did not get amplification
For negative control I agree wit Zainab.
Your gel ran very bad and because you had smear it is difficult to say what is the problem.
Contamination of your negative control. You may wanted to change your nuclease free water for your negative control and try to re-run your PCR.
Also, try to design your primers and make sure the annealing temperature is appropriate (optimize using gradient PCR)
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