Self ligation of PCR products
s535290 at aix1.uottawa.ca
Thu Dec 18 19:49:49 EST 1997
> >1. Why did unpolished DNA with overhangs self ligate?
> >2. Is there any way to check out polishing step?
> I don't see how. Why do you need to check out polishing step anyway? The
> proof should be in the pudding...:). You have to make sure that there is
> enough free dNTP's available. I usually add a uL of 2.5mM dNTP mix to the
> PCR soup before adding the polymerase.
> Actually, the frequency of dA overhangs depends on the terminal ntd. of the
> product (C/G's?) and on the enzyme being used. Not all products from a
> reaction would have dA overhang. Secondly, even without polishing, the PCR
> products are ligatable, albeit at greatly reduced efficiencies. I believe
> that the amplicons with single ntd. overhangs, if any, essentially act as
> "blunt" molecules in a ligation rxn. (not all agree with me on this) or
> that there may be enough products without the "overhang" to afford some
> blunt ligations. I have been able to ligate amplicons directly following
> PCR, without polishing, into SmaI-digested vectors.
What about the fact that even the T vector itself can self-ligate at a low
frequency. Perhaps the A-A ligation can also ? especially if DNA
concentrations are high or there is lots of ligase in the mix ?
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