efficient blunt ligation?
popa0206 at PO-Box.McGill.CA
Sat Jun 24 12:23:55 EST 1995
I think the best temperature for T4 ligase is 16 degrees and it says that
on the packing slip that comes with the enzyme.
The phosphatasing is a good suggestion.... to prepare your vector and
insert you should gel purify and prep by gel extraction (either by a kit method...
beads or resins or by spin column packed with sterile glass wool). The phenol
chlorophorm is a bit harsh unless you figure there is some heavy duty enzyme bound
to your DNA. Usually I phenol chlorophorm before running on a preparative gel. Phenol
is not the greatest to over use as it damages the DNA and can lower your efficiency.
The main thing is even if you don't find a colony (out of say 20 that you picked) at first, don't panic!
You can plate the rest of your electroporated or calcium phosphate transformed bacteria on
several plates and probe for your colony... It only takes a few days and it can save you months
of redfining your conditions for ligation. Time is your worst enemy not the work itself... sometimes
you need to sweat a little.
Graham Dellaire Snail Mail:
Red Cross, Research
McGill University Montreal Blood Services
Faculty of Medicine 3131 Sherbrooke St. East
Div. of Experimental Medicine Montreal, QC, Canada
E-mail: popa0206 at po-box.mcgill.ca H1W 1B2
B2XE at musicb.mcgill.ca
WWW Page: http://www.medcor.mcgill.ca/EXPMED/expmed.html
Fax: (514) 525 0881
Voice: (514) 527 1501 ext 175
More information about the Methods