problem of ligation... need help!

Master of the Keys Master.of.the.Keys at wanadoo.fr
Tue Sep 29 16:23:08 EST 1998


     hello

I'm trying to clone a foreign fragment of DNA in bluescript  (with cohesive
termini) and I fail...but it should be very easy and I don't  undestand
what's happend...
I'm using the T4 DNA ligase of Appligene and I  incubate my tubes on night
at 4°C. Has somebody an idea of the best ratio  fragment/plasmid , and the
best temperature of incubation...because I have  read a lot of divergent
propositions...37°C, Room temperature, 16°C  and 4°C.....My bacteria are
very competent (10 8 cfu/pg) so I think that  the problem is really coming
from the ligation....for exemple 25 ng of my  linearilized bluescript with
ECoRI (not dephosphorilated) incubated alone  with the ligase, give just 12
blue colonies  !!!!


thanks





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