5 replies to this topic

[jonathannimal]

Hallo
Please help me. I need to cut my vector with two different sets of enzymes (xhoI & xbaI ) or (xhoI &  sacI) for double digestion.
Please tell me which one will be better in digestion and for the cloning work.

urgent

nim  

[kawaka]

It depends.

Do you have any of those enzymes with you? Are their buffers compatible?

If you don't yet have those enzymes, look into NEB or Promega catalog to see if xhoI & xbaI  or xhoI & sacI can use the same buffer. If yes, you can do a double cut in a single reaction. If not, cut the plasmid with the enzyme which reqires low salt buffer, after the cut, adjust the salts to the required concentration by the second enzyme and add the second enzyme. That is it.

[george@CASE]

I agree with kawaka. The NEB catalog is a treasure trove of information as well as the little leaflet included in each enzyme. Read them. A lot of people just rush in to an experiment and then scratch their heads when it doesnt work.

[anyluntan]

Just make sure the both enzyme you use is using the same buffer, and they are both 100 compatible.

[rajgene]

Hi,
before u put the rest sites on the primers(if u do pcr cloning) make sure tht the there are no complementary bases in the overhangs  generated after double digesting the vector with rest enzymes. if not change the enzymes. this will avoid any chance of vector recirularization after double digestion. hope u got the idea.
enjoy experimenting.

[fguilhem]

HI
according to the NEB catalog, you should use the Xba1/Xho1 mix for your digestion due to the fact tey're 100% buffer compatible (NEB buffer 2 + BSA)