Cloning Unstable Sequences
andrei.popov at bbsrc.ac.uk
Thu Nov 7 18:28:39 EST 1996
Jose E. Mejia wrote:
> Dear Jon,
> The human genomic libraries prepared in the low copy number PAC
> (P1-derived artificial chromosome) and BAC (bacterial artificial
> chromosome) vectors are thought to be very representative. The clones are
> usually very stable, even those inclusive of long stretches of repetitive
> sequence. The E. coli strain used is DH10B, which can be obtained from
> Gibco BRL.
> Although the above vectors are used to clone fragments in the 100-200 kb
> range, smaller inserts (a few kb) are OK. There is a problem, however: in
> preparing DNA (the classical alkaline lysis protocol is fine), a large
> insert makes for the low copy number and the DNA yield, expressed in
> micrograms, may still be acceptable. This is certainly not the case with
> small inserts, specially in the BAC vector.
> Wellcome Trust Centre for Human Genetics
> University of Oxford
> jose.mejia at well.ox.ac.uk
I would back up this suggestion. A postdoc in our lab has just cloned a sequence
which was believed to be "unclonable" before. She did it in pBAC.
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