Genomic Library Construction

Eric Lader lader at ambion.com
Tue Sep 17 21:42:49 EST 1996


In article <malexe-1709961103560001 at 128.249.210.32> Mikhail Alexeyev,
malexe at biost1.thi.tmc.edu writes:
>The way I understand it, for your statement to be true more then
>approximately 1/3 (by mass) of EcoRI fragments in human genomic DNA digest
>should be in 9 kb range. Any data to support your opinion?
             

Nope, I was up too late last night. Thanks for caring :-) 

Anyway, His idea of a mini library hasmerit under certain situations. For
example, if there are several (or many) cross hybridizing fragments in a
genomic digest and he is intent on cloning one of them in particular. In
this case, screening a whole library would mean having to sort out which
of the many positive clones is the one he was after. If he was to run a
prep gel or sucrose gradient to select a small fraction containing only
the fragment he was after, he would be ahead of the game. He could
probably select a small enough fraction to represent a decent enrichment.
Maybe as a small improvement he could find some other restriction enzyme
that does not cut the fragment and then first isolate the fragment and
then cut with the other enzyme. This will reduce the number of ligatable
fragments.

I cloned the ends of a transgenic insertion like this, avoiding the 50
copies in the genome and targeting the novel sized junction fragments. It
wasn't pretty, but it worked.



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