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The weirdest PCR ever!


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#1 kga1978

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Posted 09 December 2004 - 09:35 AM

Hi All,

I am currently trying to PCRing out a 189 bp fragment from a plasmid. I know that the primers work correctly, but when trying to PCR out the fragment I get a 8kb (!!) band instead?!!

The elongation is only 1min, and the annealing is 30 sec, so this should be impossible!

The enzyme used is a new blend called KOD, and they say it can process about 1kb in 35sec - fast but still way too slow to get a 8kb band in a minute!

All the negative controls are negative.

I do not have the exact sequence of the plasmid - I received it from another university and they say it contains my fragment of interest.

So.... any thoughts? I really REALLY don't get this - 8kb, one minute?! Nooo way..

#2 beena

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Posted 09 December 2004 - 11:45 AM

hii
change the DNA pol. that u are using and infact since its only 189bp, u can just use Taq (it is definitely error free for this length).
reduce the extension time to as low as possible.
reg. the 8kb mystery, what is the size of the parent plasmid, is it that by any chance u have the primers such that they they are amplifying the whole DNA, s
NOTE: i find at the NOVAGEN site that KOD pol has an extension rate of 120bp/s.
kb

#3 pcrman

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Posted 09 December 2004 - 12:20 PM

Is the 8kb band the original template plasmid instead of an amplicon?

It is also possible that the extension goes on all around the plasmid before another primer anneals to the extension product if the extension rate is fast enough.

#4 kga1978

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Posted 17 December 2004 - 08:27 PM

I actually tried changing pol right after writing the post, and using Taq or Pfu I don't get the product. So it must simply be KOD amplifying the entire plasmid - which means that I probably have an antisense construct??




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