problem of ligation... need help!
Master of the Keys
Master.of.the.Keys at wanadoo.fr
Tue Sep 29 16:23:08 EST 1998
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
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 !!!!
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