I am performing PCR for my cloning , In all isolates i am getting multiple bands after the amplification .
what could be the reason.???
Try optimizing the annealing temperature.
BLAST your primers to make sure they are specific to just your gene-of-interest and not matching to several related regions.
What size band were you expecting and what sizes are you seeing?
it's better that you redesign your primers (first base pair of the forward primer should be G/C.
Try to increase the annealing temperature.
If you are also getting the expected band then you should cut that band from the gel and do the gel extraction to confirm it.
I hope it will help.
Hi, may be it is due to shearing of DNA. First you can increase the annealing temperature, and check, if problem not resolved, then you can re-extract the DNA, make fresh PCR primers and reagents.
Another thing you can do, You can try some specific primers according to your samples.
maybe due to not optimal conditions/protocol.
I would propose to revise both carefully.
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