algal DNA isolation

dunahayt at TCPLINK.NREL.GOV dunahayt at TCPLINK.NREL.GOV
Fri Apr 15 13:09:34 EST 1994


Hi,
I sent this message yesterday, but it didn't show up at my address so I 
think it got lost in E-mail hyperspace. (If it shows up at your address 
twice, I apologize).  

I'm trying to isolate small amounts of total DNA from a large number of 
isolates of an alga for analysis by Southern hybridization and PCR.  We've 
been using a "miniprep" adapted from a Chlamydomonas protocol we got from 
the Chlamy Gopher; basically a SDS/phenol/chloroform extraction.  We get 
good yields, but the DNA digests very poorly with restriction enzymes, 
presumably due to inhibition by carbohydrates.  CTAB precipitation will get 
rid of the carbohydrates, but the protocol is a pain for numerous small 
samples, and the yield is very low.  My questions: does anyone know of an 
easy protocol or a commerial product (i.e., a spin column) that will get 
rid of carbohydrate from a DNA prep?  Even better, would be a quick kit or 
something that yields digestable DNA without phenol/chloroform extractions. 
I spoke to someone about using th Puregene isolation kit (involves a 
protein precipitation instead of PCI), but I'm not sure it will help with 
the carbohydrate.

Thanks for your help.

Terri Dunahay
National Renewable Energy Lab
Golden, CO
e-mail: dunahayt at tcplink.nrel.gov



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