(by hzhen from freeuk.com)
Sat Jun 30 12:54:16 EST 2007
> 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:
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
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.
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