S. pombe BAC library

Preparation of S. pombe 972h- BAC library

A bacterial artificial chromosome (BAC) library of average insert size 88 kb was constructed in the vector pBACe3.6 (Frengen et al., Genomics 58: 250-253) at the Sanger Centre by Mike Quail, according to published methodology (Osoegawa et al., Genomics 52: 1-8).

pBACe3.6 plasmid map

Diagram from http://www.chori.org/bacpac/

Briefly, DNA plugs were made by mixing mid-log phase S. pombe cells with Incert agarose (BMA) to a final cell density of 5 x 108 cells/ml, incubating with lyticase and then proteinase K.

Ten plugs were partially digested, each with 2 units of Sau3AI, at 37°C for 20 minutes. Digests were terminated with EDTA and proteinase K, and the plugs electrophoresed according to the

pseudo-double sizing procedure described in (Osoegawa et al., op. cit.).

A gel slice corresponding to 80-100kb was excised from this gel and DNA recovered by electroelution. DNA was dialysed versus 0.5 x TE and concentrated against 0.5 x TE containing 30% PEG-8000, prior to ligation at a 10:1 vector:insert ratio with BamHI-digested and dephosphorylated pBACe3.6.

This gave a library of 80,000 recombinants of average insert size 88 kb.

Colonies were picked into 36 x 96 well plates, cultured and replicated into glycerol storage medium.

This set of 3456 clones representing 22x genome coverage were end-sequenced according to methods then in use at the Sanger, and were also gridded in a 6x6x96 grid on a single filter to enable probing.