pcr prob

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Sun Feb 24 08:40:53 EST 2008


harold b wrote:
> Hi, my negative control (no template with both primers and mastermix)
> consistently shows a stronger single band whereas my amplicons show a
> leading similar but weaker band followed by a fading smear reminiscent
> of non-amplified DNA. In each case the product with DNA is less
> intense. Can someone make sense of this?

The strong band is most likely just your primers.  Easy to check by 
running the same amount of primers used on the gel.  The weaker band is 
most likely the desired PCR product, check the size of the band (run 
marker), the smear may be non-specific amplification products.  If the 
weaker band is the right size, then you got what you wanted, you can 
just cut it out if you want to use it for cloning if that is what you 
want to do.

If you want to use the PCR  for pretty picture, then think about 
increasing the number of cycles, changing (most probably by increasing) 
the annealing temperature, changing the Mg++ concentration, lowering the 
amount of primers, etc.   Don't bother with redesigning the primer 
unless you got the wrong PCR product.




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