problem with unspecific PCR product - (Oct/18/2007 )
hi to all,
recently i did a PCR screening to check either my insert was successfully ligated into my vector. for the 1st stage of PCR screening i was using the PCR master mix by promega.i got good result and the expected band was clear.later due to the master mix was finishing, for the rest of my samples i performed PCR using PCR pre-mix.as the result, i get several unspecific band the are smaller in size compare to my expected band for each sample that i run. the unspecific band is about 100-200 bp in size, can this considered as primer dimer?there were also a few bands with the sizes higher than 100/200bp.if it is not primer dimer, what is the problem here and how can i overcome this problem?
It is likely that the small bands you see are in fact primer dimer. To get rid of the larger, non-specific products I would suggest the first thing you try is raising the annealing temperature.
i had performed a gradient PCR before this where the annealing temperature ranging from 51,52,53,....67,68,69, 70 degree.the result is still the same. unspecific bands appeared together with the suspected primer dimer band for every single different temperature.
Try running a negative control, meaning that you don't add any plasmid. Your PCR components may be contaminated. Hope this helps.