Cloning problems

Chris Yost cyost at acs.ucalgary.ca
Mon Apr 24 11:50:24 EST 1995


In article <3n62hp$dag at news.univ-rennes1.fr>, Onno Myriam at univ-rennes1.fr
(onno Myriam) wrote:

> I have given up  to use TA cloning kit to clone my PCR products because
of a lot of false white colonies.
> I try an other protocol as follow:
> 
> - Dephosphorylation of SK+ vector digested with ECORV with CIP
> - Control of the dephosphorylation after ligation, on an agarose gel: OK
> - Transformation of XL1Blue bacteria with
>                 1° insert (700 bp) +vector (ratio 1:1) ( 25 ng vector)
>                 2° dephosphorylated vector ( 25 ng vector)
>                 3° supercoiled vector (100 pg)
> 
>         Here are the results:
> 
> 
>                 1° 308 blue and 121 white colonies 
>                 2° 244 blue and 240 white colonies
>                 3° 404 blue and 0 white colonies
> 
> My questions are
>         how can I to improve dephosphorylation ?
>         may I have to modify the ratio insert-vector ?
>         why is there white colonies after ligation of a dephosphorylated
vector ?

You might want to try blunting the PCR product with Klenow or T4 DNA
polymerase to improve your efficiency. Even though TA cloning doesn't work
too well, PCR products do have an A tail that need to be dealt with one
way or another. Ligating even a 1 bp overhang into a blunt ended site
(EcoRV) is inefficient. The other thing to do is screen pools of plasmids
(ie do 24 preps on groups of 10) to bring the amount of work down a bit.

Your questions:
1. Dephosphorylation may not be that bad - there are lots of different
DNA's in a PCR reaction that could potentially ligate in. Somebody else
mentioned it may simply be concatamerization of the vector, but since they
are both dephosphorylated (ie 2 vectors), this shouldn't occur.
2. You should have a higher amount of insert (2:1 molar at least)
3. See #1.

Hope this helps.

Julian

Cellular, Molecular, and Microbial Biology
University of Calgary



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