How Much Ligase Do I Need?

ChenHA hzhen at freeuk.com
Mon Aug 21 12:13:14 EST 2006


On 19 Aug 2006 14:29:07 -0700, htert2020 at yahoo.com wrote:

>
>Thus, the initial concerns would be, would a "typical" amount of ligase
>be sufficient, and if not, how much ligase would be effective?  Would I
>have to double, triple, or quadruple the amount of ligase "typically"
>used in a "typical" experiment, or would I have to multiply the amount
>of ligase by a factor of many hundreds?  If hundreds, that can be
>extremely expensive.
>
>But, as mentioned in my previous message, I suspect that roughly the
>same (or slightly more) ligase would be needed than the "typical"
>amount because, although the concentration of DNA endpoints is super
>high, the frequency at which the ligase molecule meets a DNA endpoint
>for ligation should also be super high -- resulting in roughly the same
>reaction time as a "typical" experiment.
>
>Am I right?  I've searched many places for the answer to this question,
>but could never find it.  Perhaps you or someone here can give some
>insight based on personal experiences and knowledge.
>

A couople of  bits of information from Maniatis -

1)  condensing agent like PEG and hexamine cobalt chloride increases
ligation reaction by 3 orders of magnitude.  These reagents
concentrate the DNA  so you get very high concentration of end and
this gives you an indication of what you might expect for your
reaction.  However I think hexaminecobalt chloride tends to promote
intermolecular reaction so you get concatemers.

2) For sealing nick (which is I think what you are doing) do the
ligation reaction at 37 deg C.  (I'm never quite sure why the optimum
temperature for ligase is quoted at 25 deg C, anyone has the original
paper where this comes from?)

As for the intermolecular reaction vs intramolecular reaction, I think
it depends on whether one end is more likely to meet to its other end
or the ends of another molecule, and that is a function of the
concentration and flexibility of your DNA.  Since your oligo is short,
then perhaps high concentration doesn't matter, but I have really no
idea.  



>Thanks for the tip.  I can certainly live with slightly less ligase at
>longer reaction times.  But again, the major concern here is, "a factor
>of hundreds of times".  If I had to use 2-3 times more ligase, even 10
>times more ligase, I'd be happy.  But hundreds of times more ligase?
>That's just too much, and will become a major problem.  If I conduct
>such an expensive experiment many times in the future, that would cost
>me thousands of dollars.
>
>I happen to like NEB and am very impressed with them.
>
>Andrew



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