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bands are not discrete in PCR, why?

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#1 yobou



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Posted 30 May 2018 - 05:20 PM

Dear all

I would greatly appreciate if you let me know why bands do not appear discrete after electrophoresis of PCR reaction products? they appear diffuse and broad (as in the attached file). agarose electrophoresis was done in 2% gel conc. at 50 volts (minigel) in TBE buffer.


#2 OldCloner



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Posted 23 July 2018 - 10:17 AM

A general answer, since there is very little information here, and the "attachment" seems to be missing, is that the reactions are not well optimized.  You probably need to play with the annealing temperature or extension time. There is also the simple possibility that you are running too much of your reaction on the gel, causing the band to smear. Information that would help us answer your question include: What size band were you expecting, are you trying new primers or ones that have been used successfully before, are they degenerate, what is your template source? What is the volume of the rxn and how much do you put on the gel?

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