Help for cloning

Saengtong Pongjareankit jae at mo.mahidol.ac.th
Thu Jun 18 20:56:35 EST 1998


>

Dear Li:

I do not think that the clone grown on the plate will be self ligate of
NotI/EcoRI site.
It might indicate that you did not have a complete digestion of the vector.
You had better check the two site is far enough from each other to provide the
digestion.

Jongrak

> Help for cloning
>
> l4wang (l4wang at sciborg.uwaterloo)
> Wed, 17 Jun 1998 11:10:02 -0400
>
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> To: methods at net.bio.net
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> From: l4wang <l4wang at sciborg.uwaterloo>
>
> Subject: Help for cloning
>
> Date: Wed, 17 Jun 1998 11:10:02 -0400
>
> 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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