Help yeast genomic DNA library construction

John Thompson jrt at home.com
Sat Oct 10 20:57:32 EST 1998


In my experience, DNA purified from agarose gels contains
transformation poisons.  I once demonstrated this by gel purifying
covalent closed circular plasmid.  The gel-purified DNA yielded
100-1000X fewer transformants/ug (depending on isolation technique).

You'll get higher cloning efficiency if you purify your genomic
digest on sucrose gradients and just analyze small aliquots to find
the desired size fraction.  If you can, avoid gel purification of the
vector also, so much the better.

Regards,
John Thompson

Gianluca Molla <molla at imiucca.csi.unimi.it> wrote:

>Hello to everyone.
>Is the first time I write to this NG although I read
>the posts for more than one year.
>In our lab we are trying to construct a genomic library
>from a yeast (R. gracilis). We extract genomic DNA,
>digest it with EcoRI, run the digestion fragments on an
>agarose gel, extract the DNA from 7.5 to 9 kb from the gel (this is the
>size of fragments we are interested in) and ligate them to a cloning
>vector. The number of recombinants is very low (20-30).
>If possible we don't want to use phages. So, someone has
>experience in this kind of project? Do you think that the dimension
>of the insert (7.5-9 kb) is too large to obtain a significant number
>of recombinant? Do you think is a limit of the strategy itself or
>we can improve our results?
>Thank you for your attention.




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