phage DNA

Ken soderstrom at psy.fsu.edu
Wed Jun 9 16:32:06 EST 1999


You might consider trying a few ul of your library as a template - no
preparation required.  I routinely (and often successfully) use gt11
libraries as a template for PCR to amplify fragments up to ~ 1 kb.  I
recommend starting with 5% of your PCR reaction volume.  I don't deviate
from a standard hot-start of 94C for 30 sec prior to the first cycle.

Another sometimes effective way to combine PCR and lambda libraries is to
design primers flanking the cloning site and to use them individually with
complementary specific primers.  This will occasionally result in the
amplification of a large chunk of your gene of interest without resorting to
RACE.

Of course the better the library, and probably the better represented your
sequence is, the better these things work.

Good luck,

Ken

Adrian Clarke <Adrian.Clarke at plantphys.umu.se> wrote in message
news:7jleq7$7cg$1 at hudsucker.umdc.umu.se...
> Hi all,
>
> I'm trying to find a simple method for extracting phage DNA from lysates.
I
> wish to PCR a gene fragment from a phage cDNA library, and I was hoping it
> would be relatively simple once I separate coat proteins from DNA. Any
> advice or handy tips would be appreciated. Thanks in advance,
>
> Adrian
>
> --
> ------------------------------
> Dr. Adrian K. Clarke
> Department of Plant Physiology
> University of Umeå
> 901 87 Umeå
> Sweden
>
> Tel: +46 90 7865209
> Fax: +46 90 7866676
> Email: Adrian.Clarke at plantphys.umu.se
>
>
>





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