Help for cloning

l4wang l4wang at sciborg.uwaterloo
Wed Jun 17 10:10:02 EST 1998


Hi, all:
I am cloning a cDNA into His tag fusion protein vector (Invitrogen). No
matter how I try,   the target cDNA won't get into vector. The strategy
for the cloning is very straight forward, double digested (NotI/EcoRI)
the target cDNA and the vector as well, used 1 vector to 4 insert DNA
molar ratio to ligate. After transformation, only a few colony showed
up. All of them were vector shelf-ligation. I did a lot cloning before
and never had such experience. I thought it might toxic protein. If it
do so, what fusion protein system I could use? Any suggestion will be
greatly appreciated.


Li




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