EcoR1-Pst1 digest for vector and insert - (Feb/26/2008 )
The next clone I have to make is using EcoR1(NEB) and Pst1( NEB) digestion of both vector and insert (PCR). I was wondering if anyone could suggest a good way of doing this----I know Pst1 uses buffer 3+BSA and Ecor1 has 100% activity in all 4 buffers along with it's own buffer----I was also worried about star activity of Ecor1.
We routinely (very very routinely) do double digests with EcoRI and PstI in NEB buffer 2 + BSA. These enzymes just work. Keep the total volume high, the glycerol and DNA concentrations low. 1 ug of DNA in 100 ul, 10 ul buffer 2, 1 ul BSA, with 1 ul each of the enzymes is a good place to start.
As suggested, Digest DNA in say 50ul and make sure the glycerol concentration is low. You should be fine.
Thanks Phage434 and Scolix for the info