Anyone used restriction enzyme BsaI? - (Dec/20/2006 )

If anyone has used the restriction enzyme BsaI, I would like to clarify some reaction conditions.

I have a 3bp extension beyond the BsaI recognition site:

GTT GGTCTCAGATC CACC ATG

I digest in NEB3 at 50C for 2hrs & 4hrs.
Purify digested product by spin column purification (qiagen)
Ligate to check digestion efficiency (2hrs room temp.)
Very little dimeric band seen.

I am currently digesting for 16hrs at 50C.

Any suggestions to improve digestion??

thanks

I have used this enzyme once and didnt get good digestion. But I could work with what ever got digested. Havent used it since.

The one question would b where did u place the digest for 4 hrs at 50C. Was it compeltely immersed or kept in a hot block.

This could make a difference.

Used a PCR machine that was set to hold temp. at 50C.

I am doing a BsaI-XhoI double digestion for some cloning purposes.
I am now using 16hr BsaI digested product, digest this with XhoI for 3hrs at 37C in the same NEB3 buffer.

Spin column purify the double digested product.
Use it for ligation and then transform competent DH5alpha cells.

I had left the digest on a hot block for 2 hrs as usual. But since I didnt get a complete digest I was thinking that could b the reason. But if u used a PCR block to hold temp. at 50C, then I guess the enzyme isnt the best cutter. Anyway, try ur ligation and if u get a difference in colony numbers between control and ligation, go for it.

Good Luck !!!