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PCR problems

PCR problems - Protocols and Methods Forum

PCR problems - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-04-2004, 07:35 AM
rahul.ranjan@biocon.com
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Default PCR problems




At higher PCR cycles we observe degradation in all the samples (including the blank) in the form of smear (streaks). Although at lower cycles we observe a sharp band. Higher cycles are need to obtain an amplification of 1pg DNA (genomic DNA).

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  #2  
Old 08-04-2004, 05:17 PM
Kyle Legate
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Default PCR problems

[Only registered users see links. ] wrote:
What could possibly be degrading in your blank. I think this is a larger
problem.


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  #3  
Old 08-05-2004, 07:50 PM
Jose de las Heras
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Default PCR problems


"Kyle Legate" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]...

Yep, it looks like you're getting some contaminant which has multiple
homologies (or close enough, at the annealing temperature that you use) to
your primers... with lower cycles there may not be enough non-specific
amplification to be visible on the gel... but increase the cycles and it
becomes apparent.

Non-specific amplification with high cycles is not uncommon in genomic PCRs,
by the way. Aim for the highest annealing temp you can use, and don't overdo
the number of cycles justto obtain more product. It's best to run more
reactions.

Jose


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