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[BSM]

I was looking to perform a sequential double digest with AseI and BssHII restriction enzymes to isolate my linear construct that will be used for downstream transgenic mouse applications. I need about 60ug of cut plasmid.

AseI works in NEB buffer 3 @ 37c and BssHII works best at 50c in NEB buffer 3. I was 1st going to perform a pilot run with a small amount of DNA and incubate with BssHII 1st at 50c for about 2 hours, heat inactivate at 80c for 20 minutes then add AseI after the temperature has adjusted back to 37 and incubate 2-3 hours at 37c then heat inactivate at 65c for 20 minutes. The resulting digest should give me 5 fragments, the largest being the one I need.

My question is this, what is the optimal enzyme to DNA ratio to use for a digest with 60ug plasmid and at what total volume? My plasmid concentration is at 1ug/ul. In addition, is it necessary to CIP treat?

Thanks!

Edited by BSM, 05 November 2011 - 09:25 AM.