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. 

Thanks
Philip



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