CTAB method for genomic DNA isolation from Salmonella


Fri Jan 6 10:39:31 EST 1995


In article <RANKD.1.0008BF44 at FOODSCI.PURDUE.EDU> RANKD at FOODSCI.PURDUE.EDU (Doug Rank) writes:
>Has anyone successfully used the CTAB method (as described in the "Red Book") 
>for genomic DNA isolation from Salmonella (or E.coli)?  We have been trying it 
>but have been getting precipitates after both the NaCl addition step as well 
>as after the CTAB/heating step.  Looking at the centrifuged precipitates 
>(i.e., on gels), both of these apparently have more chromosomal DNA than the 
>final supernatant that went through to the isopropanol precipitation; the 
>final precipitant also was loaded with RNA - actually, looks like a good 
>procedure for RNA isolation.  
>
>Is there some trick to this method?  According to the Red Book procedure, a 
>DNA-CTAB precipitate may form if the NaCl conc. drops below 0.5 M, and we have 
>even added more NaCl, but no luck.  Also, we have examined the pH of our 
>phenol (buffered at pH 8.0), but can't explain why the RNA is carried over?
>
>Any suggestions are welcomed.
>Thanks in advance, Doug


Hi Doug, and happy new year!

I have tried the method you described and it worked well. Maybe I have 
some suggestions. (You are speaking about the "red book". Is it the one from 
Ausubel? It was the one I used).

Firstly, it is better to use the Nacl at a concentration higher than 0.7M, and 
I usually put more. Secondly, I have noticed that it is better to increase the
volume after the concentrated Nacl addition step (by adding new NaCl 0.7M). 
This allows a better precipitation and separation from proteins and RNA.
I hope this could help you, and good luck!

Mireille



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