single band -> multiple banding in PCR - (Jul/27/2008 )
Hi,
I'm having the strangest problem with a 'new' PCR which involves a single forward and 2 different reverse primers (in seperate reactions). I have trialled the PCR (standard conditions and given to me by another lab) and ran another round with clinical isolates (+controls) and got lovely clean single bands for the ones I should have (PCR 1) and nothing for PCR2 (as expected). I reran the PCR later that day with a second batch of clinical isolates (but the same control DNA) and got multiple banding in PCR2 (still single banding in PCR1). However, I know the banding in PCR2 is non-specific as the controls lack the specific sequence targeted. I used the same PCR machine, program (95-30sec, 53-60sec, 72-30sec times 35 cycles) etc. but changed the taq. I thought that might have been the problem, so I got out a new batch in case it was a quality control issue, but still same problem. I also used a new dilution of primers, new h2o and buffer and mgcl2 in case of some cross-over contamination. It is strange because 2 PCRs worked fine (completely as expected) and then suddenly I got multiple non-specific banding which hasn't gone away yet... Has this happened to anyone? I would appreciate any suggestions on how to fix it.
Thanks!
I'm having the strangest problem with a 'new' PCR which involves a single forward and 2 different reverse primers (in seperate reactions). I have trialled the PCR (standard conditions and given to me by another lab) and ran another round with clinical isolates (+controls) and got lovely clean single bands for the ones I should have (PCR 1) and nothing for PCR2 (as expected). I reran the PCR later that day with a second batch of clinical isolates (but the same control DNA) and got multiple banding in PCR2 (still single banding in PCR1). However, I know the banding in PCR2 is non-specific as the controls lack the specific sequence targeted. I used the same PCR machine, program (95-30sec, 53-60sec, 72-30sec times 35 cycles) etc. but changed the taq. I thought that might have been the problem, so I got out a new batch in case it was a quality control issue, but still same problem. I also used a new dilution of primers, new h2o and buffer and mgcl2 in case of some cross-over contamination. It is strange because 2 PCRs worked fine (completely as expected) and then suddenly I got multiple non-specific banding which hasn't gone away yet... Has this happened to anyone? I would appreciate any suggestions on how to fix it.
Thanks!
Maybe your block isn't performing properly. How long since it was serviced? Do you get different results using the same template in different positions of the block?
I confronted the same problem while I was trying to amplify an arabidopsis MAPK. Once I got the clear band but next time i got different weak bands and so on. I tried differet blocks and changed PCR reagents but no results. Its really strange.... 