purification after digeation - (Aug/29/2007 )
i am digesting vector with Nde1 and then i am doing gel extraction of linearised vector
then proceeding to second digestion with BamH1
in this case i am getting more loss of DNA
please suggest me if any alternative for purifying after first digestion
is that dna can be purified compltely in gel extraction kit
is taht gel extraction cal eliminate all of the enzymes from DNA
plaese give suggestions
There is no need to perform the NdeI and BamHI digests seperately - just use a buffer that they are both compatible with. (e.g. NEB buffer 2). The gel extraction kit should be fine for the purification, although I find that yields of 25% are common.
purification by gel drives a high loss ratio. better is a column or matrix kit
I think to ensure not much loss, try to introduce more sample in the first place.
I usually use phenol chloroform method though.
Double digestion works too as mentioned by bitesizebio guy.