PEG removing A overhang? - low transformation efficiency (Mar/20/2008 )

Dear Bioforum members, please share your experience with me!

I am trying to clone into the pGEM T vector, but only very few cells get transformed, and most of them are blue
The chemicals should be ok, and the protocol is standard except: I am cleaning up my PCR product with PEG (15min 37°, 15min 15000rpm, 2x EtOH, resuspend in ad, looks fine on gel). Am I removing my A-overhang with the PEG? Anybody experience with that problem?

I thought of gel elution, but I have app. 10 bands and I don’t know which one I need...and even the different band contain different DNAs...

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I am only aware of freeze-thaw cycles promoting loss of A overhangs... I don't see why PEG-NaCl would...
Anyway, thanks for putting forward this methods, I think I am going to use it for myself, PCR cleaning kits are wearing me off (as well as the amount of DNA in my preps )

You should also look at your cells, are they fine ? Do you prepare them yourself ? The what is the size of the fragment you are trying to clone ?



..and now my favourite sentence : I am a bit worried about this ^^^^^... are you telling us you make a pCR yielding 10 bands ?

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thanks for your reply, nearly forgot about this post

the PEG protocol works - in my hands - better than any comercial kit I have tested so far; you do not see any primer dimers (caution- removes DNA smaller than ~ 200bp) and the DNA loss usually is neglectable. And the big advantage is that you can "concentrate" your PCR product by just resuspending it in the amount of H2O you need to get (near) to the desired concentration....works quite well; I have obtained great Seq from a 50µL PCR reaction with a yield of 3 ng/µL by resuspending it in only 10µL ddH2O
switched from promegas TA kit to a Fermentas blunt kit and get fine results now (not the ones I want, but in principle they are fine ) and yes, maybe my cells were not competent enough.



yes if I am lucky ...I am working with obligate endophytic fungi I can not get in pure culture or purify them any other way; so I extract plant material containing my fungi; but these are not the only fungi in the plant material of course; so somtimes I have to look up what is the ladder and what my PCR product. Need to seq for phylogenetic analyis but I am on the way to specific primers now

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