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Can't find a recombinant after ligating


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#1 Ahrenhase

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Posted 09 April 2013 - 12:07 PM

I'm trying to ligate a 1kb insert into a 5kb destination vector. Unfortunately, I am only able to use a single restriction site (Sal1); therefore, the cut plasmid can easily religate. I ligated insert and vector at a 3:1 ratio. I had to have screened over 60 colonies so far and not one was a recombinant (PCR and restriction digest analysis).

EDIT: And I am certain that my insert is being cut since I first put it into a topo construct.

Edited by Ahrenhase, 09 April 2013 - 12:08 PM.


#2 bob1

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Posted 09 April 2013 - 12:33 PM

Did you de-phosphorylate the vector?

#3 Ahrenhase

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Posted 09 April 2013 - 12:48 PM

No, we have CIP, but for whatever reason my PI advised against it.

#4 bob1

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Posted 09 April 2013 - 12:51 PM

Unfortunately he is a bit correct, CIP can, and often does, cause a lot of problems, but as you are using a single cut, it should at least help to prevent the plasmid re-ligating, which would tell you if there is a problem with the insert or the ligation.

#5 Ahrenhase

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Posted 09 April 2013 - 02:15 PM

I'm giving it a go, we'll see how it turns out.




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