ES Cell Targeting Protocol

Transgenic / Gene Targeting Facility

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Preparation of Targeting Vector for Electroporation

The goal is to have 50-100µg of purified, linear plasmid DNA at a final concentration of approximately 1ug/ul. 

1.    Grow and purify plasmid DNA. 

2.    Digest plasmid DNA with appropriate enzymes to linearize and to cut out vector DNA

3.    Extract DNA with pheno/chloroform, precipitate with ethanol and re-suspend in TE.

OR:

4.   Purify using DNA purification column.

5.    Re-suspend final purified, linearized DNA in TE (allow overnight at 4°, followed by vigorous 'flicking').

6.    Measure concentration at A260.

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