blunt ligation

David Micklem drm21 at mole.bio.cam.ac.uk
Sun Dec 8 11:10:53 EST 1996


In article <32AB60DB.7CF3 at unitel.co.kr>, "Chang,jihun" <j6544 at unitel.co.kr>
wrote:

>Dear bionetter.
> 
>I'm trying to ligate Hind III-digested genomic DNA into SmaI-digested 
>pBluescript SK vector.
>but, final white transformants I got were so few.
>I want many white colonies.
>Please, give me your help.

Um, er, you _did_ blunt your HindIII ends, didn't you? And phosphatase your
vector?

More to the point, why are you doing it this way?  Wouldn't sticky-ended
ligation into the (phosphatased) HindIII site of pBluescript be a bit
easier.....

In any case, more details are needed:  do you get lots of blue colonies? 
What is the ratio of blue to white?  What about on the vector-only control?
Are your competent cells any good (uncut vector control)?

David



David

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D.R.Micklem,
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