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