Total genomic DNA was extracted using either a standard phenol-chlorophorm
method (Sambrook et al, 1989) of with a method with NaCl.
In this case, mix
50-100 ml of freezed erythrocytes of penguins twins were washed with 500ml
cold 0,9% NaCl and separated at 4500g for 5 minutes at 4oC. The supernatant
was removed by aspiration and the pellet was suspended in 1 ml buffer (10mM
tris-HCl; 0,15M NaCl, 2mM EDTA, pH 7,6). Probe was incubated in the presence
of 1% SDS and 100 mkg proteinase K at 37oC overnight. Cell's lysate, after
addition of 5M NaCl up to 1 M, was gently inverted and centrifuged at 4500g
for 20 minutes at room temperature. The supernatant was mixed with 0,7 volume
ice-cold isopropanol and DNA was spooled at glass stick. The DNA samples were
washed with cold 70o ethanol, air-dried and then dissolved in
H2O. The DNA
concentration was determined at spectrophotometer as well as with
electrophoresis in agarose gel with DNA standard.
An authors express one's thanks to the INTAS for support of this research (INTAS project 01-0517).
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