Cosmid library-Making glycerol stocks

Thursday, December 04, 2003

Description
The library is a L. pennellii library. The cosmid library was made from MboI partially digested total genomic DNA which was size fractionated on a sucrose gradient and selected for a range of approx. 15-30 Kb. the DNA was ligated into the cosmid vector T04541. This vector is a binary vector with left and right border sequences, a NPTII gene for selction in plants. The vector has a tet marker for selction in bacteria.

Procedure
The cosmids pools were received on 140mm plates.To every plate ,LB medium was added several times until 10 ml of final volume in a tube. Each time the plate was scratched and the medium was collected into the tube.
For every pool stock glycerol was made in 50% glycerol and was kept in -80 C.
Additional glycerol stocks were prepared using 900 ml of ON bacterial cultures (LB/tet 15) and 900 ml of 50% glycerol and stored at -80 C.

DNA dot blots work but give high background. PCR analysis of the superpools works well and is also faster.

Bacteria can be grown at 5-15 mg/ml tetracyclin in LB at 37 C

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Cosmid library-Making glycerol stocks