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Isolation of rDNA plasmid from Amoebae

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.

Lysis buffer
Working Concentration  Stock  100mls
10mM EDTA  0.5M 2mls
10mM Tris pH 7.5  1M 1ml
0.6% SDS  10%  6mls

References

Clarke, C.G. and Cross, G.A.M. (1988). Circular ribosomal RNA genes are a general feature of schizopyrenid amoebae. J.Euk.Microbiol. 35; 326-329.

Hirt, B. (1967). Selective extraction of polyoma DNA from infected mouse cell culture J.Mol.Biol. 26; 365-369

 
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