fill-in and blunt-end ligation

Mari L. Shinohara Mari.L.Shinohara at dartmouth.edu
Mon May 19 18:09:52 EST 1997


Hello,

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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