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[nirupakl]

Hi all,
I have been having a peculiar problem with my primer designated as F12. The primer was designed by our team based on the nucleotide sequence data that we have already obtained using different set of primers. The F12 primer is specific to our sequence and doesnot have hairpins, selfdimers, palindromes and repeats and runs. We always do a hotstart to minimize issues due to crossdimerformation. However, we have been noticing a second band of ~230bps apart from the band of interest in all PCR combinations using this F12 forward primer but four different reverse primers. This is seen in reamplification of diluted gelelute samples too. A negative control with a primer pair(f12R) minus template also showed a band at the ~230bp region.
I have enclosed the gel photo and wish to know your valuable comments.
The primer sequence is: agcattccaggaccacca.

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[chrisbelle]

nirupakl, on Dec 9 2009, 06:30 PM, said:

Hi all,
I have been having a peculiar problem with my primer designated as F12. The primer was designed by our team based on the nucleotide sequence data that we have already obtained using different set of primers. The F12 primer is specific to our sequence and doesnot have hairpins, selfdimers, palindromes and repeats and runs. We always do a hotstart to minimize issues due to crossdimerformation. However, we have been noticing a second band of ~230bps apart from the band of interest in all PCR combinations using this F12 forward primer but four different reverse primers. This is seen in reamplification of diluted gelelute samples too. A negative control with a primer pair(f12R) minus template also showed a band at the ~230bp region.
I have enclosed the gel photo and wish to know your valuable comments.
The primer sequence is: agcattccaggaccacca.

Since even with no template you have a 230bp band (obviously not primer problem), I would bet my money that your reagents/water is contaminated with something. Replace all reagents and water and run again? Re-dilute you primers. Also possibly your water for dissolving the original stock primers may be contaminated.

Chris