BamHI and SacI double digest - 100% activity in NEBuffer 4 but why not recommended? (Sep/30/2010 )
I'm digesting my DNA with BamHI and SacI for cloning. I checked NEB double digest finder for the buffer to use. Although NEBuffer 4 gives 100% activity for both enzymes, double digest is not recommended and sequential digestion is suggested. Why is that so? Has anyone done double digest against such advice by NEB? How did it go?
I guess it's because of star activity of one of the enzymes with NEBuffer 4. NEB offers high fidelity enzymes of both REs, you may want to use (hough the costs are higher).
The costs are identical, not higher.
Yes, I did a double digest of BamH1 and Nhe1 (in Buffer 2). Not recommended, but I had no problems. I also think that star activity might be the reason for this. I'd recommend to use max 5% of your total volume of RE (ie 0.5ul of each BamH1 and Sac1 in 20ul total volume) and increase restriction time to 2hrs at 37C. Should be working.
Yup.I have conducted double digest with BamHI in buffers 2, 3,and 4 with no ill effect. I think as long as volume of BamHI is below 5% of the final digest volume, star activity can be avoided with BamHI