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Conventional PCR

PCR FAILURES

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#1 Kabogere

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Posted 23 April 2012 - 05:23 AM

I do conventional PCR for HIV single genome sequencing but for four months now my bands form in the wells. My target is 2.4kb and i have been troubleshooting to find out cause but it is not yet clear. My protocol involves a separate cDNA synthesis step. Could any one be facing the same problem or have ant possible solutions to this? Thanks...Kabogere

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#2 Tabaluga

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Posted 23 April 2012 - 09:37 AM

It could be the result of over-amplification,i.e. too much PCR product has been built (and subsequently loaded on the gel).

Here's a thread where someone has described a similar problem:
http://www.protocol-...posts/5351.html

Also, this link might help: http://palumbi.stanf...FoolsMaster.pdf
see p.12 "Bright bands in well of gel"

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 





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