nonspecific band at 50bp in PCR - (Sep/27/2012 )
Hello ...
I m working with five sets of ssr primer which i have tested earlier and i got bands at 300 bp. but now i am using same set of primers, same reaction mixture and same dna but i m getting faint bands at 50 bp..please help..please
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.
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.
kyakhoob on Fri Sep 28 12:03:29 2012 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...
hobglobin on Fri Sep 28 13:41:10 2012 said:
kyakhoob on Fri Sep 28 12:03:29 2012 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...
and the DNA was ok? and I didn't get what centrifuging primer working solution has to do with thawing? I store my working solutions in the fridge and centrifuge them shortly before using, to get down the condensed water and mix it. The same with other solutions from the fridge where this problem may occur.
hobglobin on Tue Oct 9 15:52:23 2012 said:
and the DNA was ok? and I didn't get what centrifuging primer working solution has to do with thawing? I store my working solutions in the fridge and centrifuge them shortly before using, to get down the condensed water and mix it. The same with other solutions from the fridge where this problem may occur.
'hobglobin
yes my DNA is ok. As you said that that "your
well I actually do it when they were quite long in the fridge, because when using them frequently, the condensing water is no problem...anyway with frozen and then thawed solutions mixing is more important to avoid heterogeneous concentrations within the tube...especially with MgCl2 and buffer....and after this, the centrifuging is not necessary of course.
hobglobin on Wed Oct 10 16:32:57 2012 said:
well I actually do it when they were quite long in the fridge, because when using them frequently, the condensing water is no problem...anyway with frozen and then thawed solutions mixing is more important to avoid heterogeneous concentrations within the tube...especially with MgCl2 and buffer....and after this, the centrifuging is not necessary of course.
Ok..I am going to try it again..... fingers crossed ...
