Kill mouse by cervical luxation and dissect liver. Add to a 50ml tube and place on liquid nitrogen. Grind to a powder under liquid nitrogen using a mortar and pestle.

Add powder to 10 volumes of extraction buffer:

Extraction buffer:  Mix by inversion several times and incubate at 37℃ for 1hr. Note on all subsequent steps avoid shearing. Do not vortex, shake or pipette the solution through narrow gauge pipettes.

Add proteinase K to 100µg/ml and mix in with a glass rod. Incubate at 50℃ for 3hr.

Extract 3 times with phenol saturated with 0.5M Tris pH 8.0, 10mM EDTA. Each extraction should be mixed for 15min at RT slowly on wheel, spun 3K in benchtop for 10min and the supernatant removed with a large bore pipette.

Extract twice with CHCl₃ saturated with 0.5M Tris pH 8.0, 10mM EDTA.

Dialyse aqueous phase against three changes of 1000ml 0.2 X SSC for 24hr.

Precipitate DNA with 0.1 volume of 3M Na acetate, 1mM EDTA pH 7.0 and 0.54 volumes of isopropanol. Spool DNA on a glass rod and dissolve in 10-20ml 0.2X SSC overnight.

Read OD_260-280 and set up digests of 10µg in restriction buffer with and without EcoR1. Incubate overnight at 37℃. A continuous smear, with a prominent satellite band around 1kb, with EcoR1 and a tight band at limit mobility (>23Kb) without the enzyme, should be apparent.

 Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au

David Bowtell PMCI October 1998