yeast chromosomal DNA digest
ANDREI.POPOV at bbsrc.ac.uk
Thu Aug 31 10:39:39 EST 1995
>I'm not sure if anybody else has this problem but I seem to be unable to
>efficiently digest yeast chromosomal DNA with EcoRI.I'm using the
>recommended buffer and a large digestion volume of 200ul.The DNA is
>purified using a lyticase protocol with many phenol steps.Does anyone
>have experience with this? Thanks, Paul Bundock.
you do not need any phenol steps.
Just grow yeast in 10 ml culture, wash 50 mM EDTA,
spheroplast in 1 ml SPEM (SCEM), make 1% agarose blocks (2-3),
lyse O/N in 15 ml YLS (1% SDS, 100mM EDTA, 30 mM 2-ME).
Washe twice with TE, equilibrate with EcoRI buffer and digest
1/4-1/2 of a block with 50-100 U of enzyme in 100 mkl.
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