Help for cloning

Roger Fachini rfachini at amgen.com
Wed Jun 17 13:58:07 EST 1998


Hi Li,

You could try dephosphorylating the vector with alkaline phosphatase to
lower the self-ligating background.  

In article <3587DC49.77963829 at sciborg.uwaterloo>, l4wang
<l4wang at sciborg.uwaterloo> wrote:

> 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

-- 
Roger Fachini
Bacterial Expression
Amgen Research

The opinions expressed are solely the user's and not those of Amgen.



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