Posted 20 January 2005 - 07:41 PM
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.
Posted 20 January 2005 - 11:04 PM
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.
Posted 22 January 2005 - 09:14 PM
Posted 30 January 2005 - 08:18 PM
Posted 07 February 2005 - 09:50 AM
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.
Posted 07 February 2005 - 10:28 AM
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)