Colony screening

clarosa clarosa at biocomp.unl.edu
Tue Jun 12 15:24:01 EST 2001


It is difficult to quess without knowing your pcr conditions and the oligo
sequences, but I speculate that your gene specific primers are priming some
of the genomic sequences and are effectively removed from forming the
desired product.

Bertrand Collet wrote:

> Hi everybody,
>
> Several people from our group noticed a unexplained phenomenom:
>
> When doing TA cloning, we firstly screen the colonies by PCR. Most of
> the time, when we use the 2 specific PCR-primers it does not work but
> when we use one plasmid specific primer plus a specific PCR-primer it
> works beautifully. Is there anybody who noticed this before ? What's
> behind it ?
>
> I noticed this in PCR products from 200 bp to 2 Kb, and each time the
> sequence is fine but the PCR on the colonies works only if you use 1 or
> 2 plasmid primer.
>
> Thanks in advance for your explanations,
>
> Bertrand
> --
> Dr Bertrand Collet
> Fish immunology group
> Zoology Department
> Tillydrone avenue
> Aberdeen AB24 2TZ
> Scotland - UK
>
> Tel (office): (+44)(0)(1224)27 3796
> Tel (lab): (+44)(0)(1224)27 2870
> Fax: (+44)(0)(1224)27 2396




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