BamH1-Pst1 digest - (Feb/08/2008 )

Hi everyone
I have to do a BamH1-Pst1 digest and was wondering if anyone could recommend a good way to do it
thanks
Lisa

1. Look at the sequence. Find out how many basepair separate the BamHI and PstI restriction site.

2. If the number of basepair exceed 6bp, conduct a double digest can be conducted.
If less, conduct a sequential digest. PstI being the first enzyme to use. Once DNA is linearised follow up with BamHI

3. Conduct digest in NEB buffer 3 (or similiar) using the appropriate concentration of Buffer, BSA and enzyme. The total volume of enzyme added should not exceed 5% of the digest's volume. Digest for the time appropriate to the quantity of DNA being cut. Digest volume should not be less then 20ul to avoid any evaporation problems.

i would go for a pst1 overnight digestion, and in the morning add BamH1 enzyme for 1-3h without changing the buffer