Cloning Unstable Sequences

Jose E. Mejia jose.mejia at well.ox.ac.uk
Wed Nov 6 15:32:14 EST 1996


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.

This book chapter by the people who developed the PAC cloning system may
be useful:

Ioannou, P. A. and de Jong, P. J.: Construction of bacterial artificial
chromosome libraries using the modified P1 (PAC) system. In N. C.
Dracopoli et al. (eds): Current protocols in human genetics, pp.
5.15.1-5.15.24, John Wiley & Sons, New York, 1996

Best wishes,

Jose

Wellcome Trust Centre for Human Genetics
University of Oxford
jose.mejia at well.ox.ac.uk



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