Posted 24 February 2005 - 09:35 AM
Well in theory the vector will still be able to religate. I am a bit bevildered to see that ppl say that having either two different sticky ends or one sticky plus one blunt, could not result in vector ligation. This is infact not true, since TWO vectors would be able to religate, i.e. the two vectors would "twist" into ligating to each other. Infact this happens quite often.
The dephosporylation of the vector is important in some cases but not always required - I will however suggest you always to do this, and only skip the step if you have no positive colonies growing. Also the dephosporylation is pretty easy, since it can actually be done in the restriction buffer - if you use CIP you will have to add a little bit of zinc ions first though (I usually add a bit of the CIP 10x buffer to my restriction reaction).
Okay - and to your last question. The ligation requires a phosphoryl group in order to ligate two pieces of DNA (just think of the backbone structure of DNA). If you where to dephosphorylate the vector these missing phosphate groups would be missing and (in theory) vector religation or vector-vector ligation shouldn't occur.
So when you do the actual ligation reaction with product + vector, your product would provide 2 phosphate groups (one for each strand), but since your vector is not able to provide two as well, you will actually get "single-stranded" DNA ligation - i.e. your ligation product would have two nicks - one in each end. Usually this is not a problem however, since the bug machinery will quickly fix this when you transform them. However, probably due to this fact, you will get fewer colonies when dephosphorylating your vector.....
Also don't waste time on trying to digest your ligation product to see if you have the right thing - it seldom works. Just stick the thing into your bugs, pick 3-20 colonies and do a digestion (i.e. don't waste time on a PCR either - they result in billions of false positives).
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