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!
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