genomic DNA from marine invertebrates

Sven Vilim vilim at inka.mssm.edu
Sat Aug 5 19:12:23 EST 2006


I have used this method on Aplysia Californica, it should work on other 
marine invertebrates (I am not sure about crustaceans - though it should 
work after disruption of their exoskeletons).

1) wash with 70% ethanol to remove salts - I would disrupt the 
exoskeletons of the crustaceans at this stage.

2) wash with 95% ethanol to ppt nucleic acids, proteins and extract 
nonpolar molecules such as pigments.

3) wash with TBS with 1mM EDTA to rehydrate and remove any small water 
soluble molecules.  Inclusion of RNAse A in the washes can be used to 
liberate RNA from the tissue (sample volume determines duration volume 
and number of washes).

4) Liberate DNA by digestion with Proteinase K (as per Maniatis - 
duration again depends on sample volume) digest until tissue dissolves.

5) Phenol CCL3 extract, CCL3 extract and ethanol ppt the DNA. 
Alternatively, you can feed the digested samples into any of the genomic 
DNA isolation kits/protocols.


It should also be possible to adapt the procedure to a 96 well format. 
The key is to use the ethanol and TBS washes to remove the various 
inhibitors.

Good Luck,

Sven
 ><><><><><><><><><><><><><><><><><><><><><><
     Ferdinand Sven Vilim PhD
     Associate Professor
     Department of Neuroscience (Box 1065)
     Mount Sinai School of Medicine
     One Gustave Levy Place,  New York, NY 10029
     Phone: 212-241-5981    Fax: 212-860-3369
     email: vilimNOSPAM at NOSPAMinka.mssm.edu
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