Help yeast genomic DNA library construction

Dr. Duncan Clark duncan at nospam.demon.co.uk
Fri Oct 9 03:02:47 EST 1998


In article <3618E7D9.CE2E7938 at imiucca.csi.unimi.it>, Gianluca Molla
<molla at imiucca.csi.unimi.it> writes
>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).

Check out each step of the procedure with appropriate controls i.e.

Can you re-ligate on it's own the EcoR I cut genomic?

Can you re-ligate after gel purification?

Can you re-ligate your EcoR I cut vector prior to phosphatasing?

Do you phosphatase your vector - does it re-ligate? (It shouldn't!)

Can you clone any other EcoR I fragment into your vector

Are you trying to make the library in E.coli or yeast?

What is your transformation frequency - good, bad?

What is the vector you are using? 

For a representative plasmid gene bank of yeast you are going to need a
very high transformation frequency/electroporation frequency and a lot
of recombinants. That is why a lambda library may be better because you
can get both conditions relatively easily.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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