Double restriction digest

[Steven Kirov]

Hi Rick,
If you can separate the nicked from the double cut vector all you have to 
do is to gel purify the vector. This is possible if the distance between 
the two sites is about 5-10% of the size of the vector. If this is not 
the case you can do something else: cut with the first enzyme, check if 
it is OK, then cut it with the next, but setting up another reaction with 
uncut vector and the second enzyme in the same buffer to see if it's 
working. Be carefull, some enzymes don't cut very well at the end of a 
DNA fragment and you should use more enzyme! I think NEB has a chart in 
their catalog. And of course at the end again gel purifiy the vector or 
if the small fragment is less than 30-50 bps you can use spun column 
chromatography (Sephacryl S-400).
Best regards

Steven Kirov
PhD student
Dept. of Biochemistry
Medical Faculty
Sofia
Bulgaria