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Strange problem with digestion and dephosporylation of PCR product


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#1 Dante_D

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Posted 19 January 2011 - 09:00 AM

Hello everybody,
To get 2 PCR products I used two sets of primers with BamHI and Not1 sites and I added extra 6 nucleotides to each primer. After PCR I got my two products exactly the size I wanted (+-1000bp) and no other products. When I digested them with BamH1 and Not1 (1hr, 37 celsius degrees, buffer suitable for both), dephosphorylated them (CIP phosphatase) and purified on gel I got two bands for both products- one the same size as PCR products and one about 50-100bg bigger... Does anyone have any idea? I purified both products and I tried to ligate them twice but my ligations didn't work (I didn't get any colonies).

#2 ElHo

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Posted 20 January 2011 - 02:23 AM

Dephosphorylation is usually performed on vectors rather than inserts. If your vector also was phosphatase-treated, the fragments cannot ligate. Did you perform any controls?

#3 perneseblue

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Posted 22 January 2011 - 08:41 PM

Also note that NotI requires a minimum of 9bps of overhang for efficient digestion. With only 6bp the efficiency is only 10% even after an overnight digest. So this too will be a contributing factor
May your PCR products be long, your protocols short and your boss on holiday




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