SEQanswers - View Single Post - Advice on DNA Extraction from RNAlater stored tissue

pig_raffles · 06-14-2016, 06:27 AM

Apologies for the lack of detail.

I am using fish liver tissue.

It is a tissue that I have never had too much problem carrying out extractions with before (for ethanol stored tissue). In fact the issue previously has always been too much DNA!

I have soaked the tissue prior to lysis in both water and 1 x TE, it did not seem to noticeably improve the results. Would you expect a better removal of guanidine salts with PBS?

I am pretty certain that it is co-precipitaiton of guanidine salts from the RNAlater that is the main cause of the low 260/230 ratio and also is keeping DNA bound and unaccessible - hence the low yields. Once when trying a spooling protocol (the ThermoFisher protocol to be exact), I got massive amounts of salt precipitating out. However I would be happy to be proved wrong!

06-14-2016, 06:27 AM	#3
pig_raffles Member Location: Hawaii, USA Join Date: Feb 2012 Posts: 20	Apologies for the lack of detail. I am using fish liver tissue. It is a tissue that I have never had too much problem carrying out extractions with before (for ethanol stored tissue). In fact the issue previously has always been too much DNA! I have soaked the tissue prior to lysis in both water and 1 x TE, it did not seem to noticeably improve the results. Would you expect a better removal of guanidine salts with PBS? I am pretty certain that it is co-precipitaiton of guanidine salts from the RNAlater that is the main cause of the low 260/230 ratio and also is keeping DNA bound and unaccessible - hence the low yields. Once when trying a spooling protocol (the ThermoFisher protocol to be exact), I got massive amounts of salt precipitating out. However I would be happy to be proved wrong!