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PCR for long and repetitive region from genomic DNA


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#1 thegene

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Posted 19 January 2011 - 07:00 AM

Dear mates,

Can anybody give any hints for amplifying long amplicons (14KB) in a region with very repetitive sequence from genomic DNA?

I am trying to amplify a region in the genomic wich is not annotated in the human genome data base, i know the sequence flanking this region and there i have designed my primers. I have tryed many polymerases and used betaine and DMSO with different conc and nothing worked!

Thank you so much for any suggestions

#2 thegene

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Posted 19 January 2011 - 08:53 AM

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#3 mdfenko

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Posted 19 January 2011 - 09:11 AM

do you get anything at all?

are you sure your primes don't form dimers?

if the primers are good (you can test them with internal primers for shorter fragments) then you can try using a proofreading polymerase for longer times at reduced temperature (eg 60C instead of 72C). slow and careful.

additives may not be necessary unless you have a high g-c content.
talent does what it can
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#4 SJB

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Posted 20 January 2011 - 05:01 AM

For long amplification I have had great success with Takara LA PCR kits, of 25kb and beyond.

I agree with the previous comment that the extension temperature should be kept low, experiment with that to get the one you need.

Try also designing several primers flanking your region of interest, just in case it is one of the primers that is causing trouble - despite the best design, it does happen sometimes. Primers are cheap and it saves a lot of time to order several pairs at the outset, rather than spending a long time attempting to optimise for a primer that is being difficult!

Finally, maintain positive PCR checks on your DNA template. For a long PCR you need good quality DNA, fresh preps will help with that when the positive controls tail off.

Good luck!

#5 thegene

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Posted 21 January 2011 - 12:53 AM

Thank you nso much for the hints :)




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