DNA extraction from ruminant placenta-help please!

Mr M.R. Owen mochus at liverpool.ac.uk
Fri Oct 27 04:39:26 EST 1995


I am trying to get a 2.5kb amplification product from sheep and cattle 
placental cotyledons.  I have been able to amplify a 200kb product from
simple crude lysates of the tissues (in brief, 500mg of tissue is crushed
under liquid nitrogen, then digested in 500-1000 microliters Tris/EDTA
pH 7.5,tween 20, proteinase k for 36 hours, boiled 5 minutes, and
5microlitres of supernatant is used in 100 microlitre pcrs).
Amplification of the 2.5kb fragment, which part of a multicopy gene has
been unrewarding, even after attempting phenol/chloroform extraction
from the lysates produced as above. (The primers work on pure DNA
extracted from other tissues).

If anyone would be kind enough to describe any protocols that may give me
more joy, I would be extremely grateful.

I have tried one commercial kit, which I won't name, but which was not
satisfactory.

Hoping to get some sound advice-thank you in anticipation.
martin owen
email mochus at liverpool.ac.uk 





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