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Ligation reaction - temperature and duration consideration - (Sep/01/2005 )

what could happen if the ligation reaction occur over night 37degrees when the protocol says to do it 4 overnight or 25 for 1 Hour.


The temp is low to protect the half-life of the ligase. So, the warmer it is, the quicker it will stop working. I would not suggest doing the ligation at 37oC. I do it on the bench for an hour.


maybe spike it with a bit more ligase and let it go for an hour or two at room temp.



I always do ligation 16 degree overnight. My boss said it is the perfect condition for ligation...


Whenever I have done ligation at 16C o/n I ve got better results! Even RT for 1hr should be fine i you can afford more enzyme.


thank you all smile.gif
i just did the transformation after ligation and it seems to be all right,
hoever less effective.