Sub-genomic library - difficulty with ligation
Philip Young, IWBT, University Stellenbosch
9369791 at SUN-AKAD2.SUN.AC.ZA
Mon Jul 13 16:08:41 EST 1998
Could someone who has made a sub-genomic library before please help
me by telling me what ligation conditions are optimal for this sort
of ligation (i.e. where it is important that all the fragments
in a certain order need to be cloned).
I am cloning into pBluescript, the fragments are 8000-9000 bp, and
are from plant genomic DNA. The fragments are Bgl II and the vector
is Bam HI digested (SAP-ed). I have tried several ratios but never
get more than 50 colonies (blue-white selection) per plate. I have
done minipreps of some of the "positive" clones (white ones) to
verify the size of the inserts and they are of the right order - just
too few of them. I get the "best" results (~50) if I do not SAP, but
rather digest with Bam HI and Bg lII while ligating. I am using
competent Dh5alpha E.coli cells (Rubidium Chloride method), and have
also used DH10B (yet saw no significant difference) - and am
currently attempting electroporation.
I dont know if the problem is ligation or transformation.
Could anyone out there please offer me some advice.
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