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kyakhoob

Member Since 27 Sep 2012
Offline Last Active Oct 10 2012 11:31 PM

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hobglobin
09 Oct 2012 - 07:08

#142418 nonspecific band at 50bp in PCR

Posted hobglobin on 28 September 2012 - 05:41 AM

kyakhoob, on 28 September 2012 - 04:03 AM, said:

habglobin

thank you for your suggestion actually i have tried everything. i have added every reagent properly..i tested the DNA and found OK..primers are few month old. my guide told me that the primers might get contaminated. is there any way to know whether the primers are okay or not. please help.

how did you check the DNA then? and primers, I'd also replace, i.e. throw away the working solutions and thawing new ones (my primer working solutions are frozen as several diluted aliquots from the stock).
also checking the thermal cycler is an idea, i.e. asking if colleagues have no problems with their PCRs and if your program is still untouched...

#142343 nonspecific band at 50bp in PCR

Posted hobglobin on 27 September 2012 - 06:54 AM

Sounds as if this are primer-dimers that occur at this size....i.e. your PCR did not work...perhaps one of the reagents is kaput now (often dNTPs), or DNA degraded, or you forgot to add a reagent to the reaction (Taq, dNTPs, ...)? You have to try out and use a positive control, then you know that the reagents are okay.