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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