2 replies to this topic

[kohei]

Hi,

I have previously done PCR, electrophoresis, gel excision & purification on my DNA sample and ligated some of the purified DNA into my desired vector. All's fine - plasmid extraction & sequencing.

My question is, I still have some leftover purified DNA sitting in the -20ºC freezer. Can I go ahead & do another round of ligation and/or do I have to do any prep work to increase their "efficiency". I read that it is best to do ligation when the DNA is fresh; my samples have been idling in the freezer for >1 month.

I welcome all suggestions, advice, potions etc.

Cheers.

[phage434]

What is your DNA stored in?  DNA in TE, frozen, will probably be pretty much in the same condition you started with, modulo some freeze/thaw damage.  If it it in water or (worse) with Mg++ or other metal salts present, then lots of bad things can happen.

[lizzy]

Just do it! I kept my DNA in the -20ºC freezer for about 2 month. I do a ligation with it, and the result is good.