|
This
method is a modification of the Hirt (1967) cell fractionation to
isolate the extra chromosomal plasmid that carries the ribosomal RNA
genes in Schizopyrenid amoebae (and other protists)(Clark & Cross,
1988).
1).
Harvest cells from culture by centrifugation and wash in suitable buffer
(e.g. Neff’s, or 75%
seawater).
2).
Take up cell pellet in lysis buffer (below)
3).
Slowly add 5M NaCl drop-wise with swirling to a final concentration of
1M.
4).
Incubate 4oC for as long as possible (overnight?).
5).
Centrifuge 40’, 8krpm in Ja20 rotor.
6).
Ethanol precipitate the supernatant by adding 2x volumes of cold
ethanol.
7).
Centrifuge 20’, 10krpm in Ja20 rotor.
8).
Re-suspend pellet in water, add RNAse incubate 30’ room temp.
9).
Phenol chloroform extract. |
