CTAB method for genomic DNA isolation from Salmonella

4700gbera at umbsky.cc.umb.edu 4700gbera at umbsky.cc.umb.edu
Thu Jan 5 11:37:51 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.  
[snip snip]

I've been using CTAB method for E. coli for the past month and I like it 
a lot. It does tend to get cloudy and stringy after the NaCl and CTAB steps,
presumably due to lysing of the cells. I've never gotten a precipitate, though.
After the chloroform extraction, I do get major goo at the interface. 
Is the precipitate still present after the chloroform extraction? Is it in
the chloroform phase? I also find the final product has a great deal of RNA,
I just add RNase to the reconstituting buffer. 

How much DNA are you getting? I usually end up with 2-4 ug.

-- gb





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