gene targeting in ES cells - (Oct/09/2007 )

For me to start, can anybody will share thier protocol for gene targeting on ES culture (in vitro). I have to knock out a few genes from ES cell line by means of homologous recombination (or any other method), please help.

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check this, link http://www.millipore.com/techpublications/tech1/pb0006en00
the pdf contains the protocols we use (with some little modifications).
but the problem is that I find a very low rate of homologous recombinant clones to surviving clones 1:500 and I am searching for any advise to increase the rate (in literature it may reach to 1:5)!

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