Is time of ligation insert and vector crucial? - (Jun/28/2007 )

Hi

Is time of ligation crucial. I am aware that it must take some time, but what I am concerned about is that I can't do electroporation transformation soon enough. So maybe I should stop at precipitating insert-vector mixture. Or maybe I should carry on and resuspend the pellet in water and put it, let's say, in -20.

Has anyone did that before?


Thanks

Rob

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You could resuspend the DNA in water and store it in -20C if you want o postpone ligation to a convenient time. But the time for ligation we use is quite short. So you could proceed with ligation as well.

For sticky end ligations, We leave for 15-30 min. before transforming. For blunt end, I would leave it for 1 hr.

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Thanks, but I want to postpone electroporation transformation not ligation. I know that inserts can sit in -20 or in 4 degrees for a very long time.

What I have is insert and vector ligated already. Can this mixture be stored in water or stored while being precipitated with 100% ethanol.

Thanks
Robert

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I leave the ligation mixture as it is at RT on the bench. I would leave it for 2-3 days even upto a week before transforming them.

You could store your ligation mixture as it is in 4C or -20C and use them later for transforming. I would not precipitate them as I worry that I might lose DNA while precipitating as there is too little DNA to begin with.

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i let the ligation mixture 3 to overnight if it's on the bench.
I don't precipitate anymore and just electroprate at 1800V instead of 2500.

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