Ligation

ChenHA via methods%40net.bio.net (by hzhen from freeuk.com)
Sat Jun 30 12:54:16 EST 2007


DK wrote:

> 
> Not sure where I remembered these numbers from (now I feel 
> it's for some other organism, not E.coli) but this paper shows 
> that for DH10B there is hardly any difference in per mole efficiency
> between 7 to 80 kpb (which, they say, was not true for other 
> strains), with the efficiency dropping 20-fold when going 
> from 150 to 240 kbp: 
> http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=306974&blobtype=pdf

Thanks for the reference.

As for the concentration of DNA for ligation, I think I've seen 
protocols from other people who use up to 200 ng per 10ul.  I should say 
I've never use anything close to that - I'd say 10-50 ng per reaction 
(20ul) is the usual, although I think up to 100 ng should be fine.

I don't think however there should be any problem ligation just one base 
(never done it, but regularly ligate NdeI which has a 2 base sticky 
end).  If I'm to do your ligation, I'd just leave in fridge o/n or even 
a couple of days, or high concentration of ligase at room temperature 
for a few hours (or first at 12 degC for an hour, then at higher or 
lower temperature).

I'm never quite sure about the effectiveness of using PEG or other agent 
like spermidine.  It concentrates the DNA, so when you add PEG, you are 
actually ligating at higher concentration.  I'm not sure how it avoids 
concatenating the DNA.  I'd say that it is unnessary if you do it right.

> 
> DK


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