fill-in and blunt-end ligation
Mari L. Shinohara
Mari.L.Shinohara at dartmouth.edu
Mon May 19 18:09:52 EST 1997
I posted the following message lately. A couple of people seemed to
wonder what happened after ligation: Yes, I got colonies. However,
there was no insert, or totally wrong fragments were found. If you
have a good advice, I appreciate your reply. THank you in advance.
>I have a hard time cloning. Unfortunately both fragments, insert and
>vector, did not have good restriction sites and I had to cut them with
>all different enzymes generating 5'-protruding ends. Then I
>dephosphrylated only the vector fragment first, which has the Amp gene
>there (I don't think it matters whether I dephospharylate this before
>or after filling-in), and filled both fragments with Klenow with dNTPs.
> Blunt-end (supposed to be) ligation was performed finally at room
>temperature for 2 hours with T4 ligase. --- I tried this more than 5
>time, but it really doesn't work...
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