How Much Ligase Do I Need?
hzhen at freeuk.com
Tue Aug 22 08:32:50 EST 2006
On 21 Aug 2006 22:36:35 -0700, htert2020 at yahoo.com wrote:
>> I'm just saying that there is no fixed amount of ligase as such, rather
>> how long you can leave your reaction for (just make sure there is enough
>> ATP). Is there a method for determining if your reaction is effective?
>I would say 90% successful products would be considered an effective
>ligation for the purposes of my experiment.
I suppose you might see the result in high concentration agarose gel?
>> Just something that you might consider - I read somewhere that using
>> sonication (I think it's a sonication bath) can speed up the ligation
>> reaction by making ligase detach faster from a ligated ends to another
>> unligated ends. So that a hour reaction can be done in 5 minutes or so.
>> Don't know if it will help in your case (would sonication affect the
>> way your DNA anneal? I don't know), but perhaps you can look into that.
>> I don't have the paper, but have a search, I can probably find it
>> somewhere if you can't find it.
>This is a great tip.
Some people find that you need the right type of sonication bath for
this method to work.
>What I've learned from all this is that it is best not to use too high
>a DNA concentration -- otherwise, there is an increased chance of
>concatemers and there is a chance of wearing out the ligase if there
>are too many ends to ligate.
What you can do is to anneal at a lower concentration of DNA since
this is the part that determines whether you get concatemers or not,
then concentrate the DNA for ligation. You might concentrate by
driving out the water in some ways (so you need a lower concentration
of buffer to start with), or using condensing agents or other means.
There must a formula somewhere that calculate the likelihood of inter
vs intra molecular reaction. Looked in Maniatis but can't find it.
>I've decided to keep the DNA concentration lower than I had originally
>hoped. My recent calculations show that the required ligase will still
>turn out to be affordable.
Since the activity might be much higher at high concentration of ends,
you might not need much at all. As I said before, you probably just
need to make sure that there is enough ATP (it is probably present in
great excess already, but since you are ligating a large number of
molecules, the reaction will probably slow down when the ATP gets
depleted). So perhaps the strategy would be to do the ligation
normally, then add extra ligase and ATP after some time.
>Thanks for your help.
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