Restriction enzyme digestion problem - (May/18/2007 )

Hi


I want to cut out my insrert DNA from the vector. Two restricition enzymes I can use. Nco1 which is a steaky end producer and Fsp1 which is a blunt cutter.

Can I cut the DNA at the same time with this two enzymes or I need to cut first by Fsp1 and then cut by Nco1?

plz give me a better suggestion.

I don't have any experience with FspI, thus I don't know if FspI is a good enzyme. I have used NcoI and found it to be okay. FspI works in NEB buffer 4 while NcoI apparently can work in any NEB buffer. So in theory you can do a a double digest adding both FspI and NcoI into the same digestion made up with NEB buffer 4.

Since tthis digest cuts out your insert, ie you can see a new band forming from a sucessful digest. I would say, give the double digest a try. You can easily tell that the digest has worked if you can see the band that you want.

you can always check very easily from this calculator

http://www.neb.com/nebecomm/DoubleDigestCalculator.asp

you are lucky we dont need to step by step digestion in this case