LB vs. TB?
tan at aeolus.vmsmail.ethz.ch
Tue Nov 16 09:45:47 EST 1993
In article <2c8tn6INNcn6 at emory.mathcs.emory.edu> medtjm at bimcore.emory.edu (T.
J. Murphy) writes:
>In article bloksber at thomashaw-at.css.msu.edu, bloksber at pilot.msu.edu (Leonard
N. Bloksberg) writes:
>> I have heard claims of doubling plasmid yields by growing bugs up in TB
>> rather than LB. Does anyone have any experience to support or debunk this
>> notion? Since TB is more trouble to make up, and costs a lot more, is it
>> worth it? Media recipes follow. Thanks.
>> [media recipes deleted]
>My lab started playing with TB several months ago. We now
>use it for large scale plasmid preps, but use 2XYT for minipreps.
>We no longer use LB for anything.
>TB is too rich for minipreps by alkaline lysis, the DNA from TB mini-
>cultures is contaminated with inhibitors of many restriction
>enzymes...I guess complex carbohydrates but am not really sure.
>Yes, you can get phenomenal growth and yield in TB compared to LB.
>For large scale preps, we get better than 4X what we were getting in
>LB, wheras LB gave maybe 1 mg plasmid/liter, TB is giving 4-5 mg/l.
> [stuff deleted]
I recently started using TB instead of 2xTY to grow 5 ml cultures for alkaline
lysis minipreps and I'm quite happy with the results. I don't consider this to
be a contradiction to what T.J. Murphy wrote since we could be using different
host strains and the details of our alkaline lysis minipreps might vary. For
the record, I use TG1 as host strain for most of my plasmid minipreps. My
alkaline lysis miniprep is an amalgation of various procedures: alkaline lysis
-> isopropanol ppt -> resuspend in RNase soln -> 1 x phenol/CIA extraction ->
Sepharose CL-6B or Sephacryl 400 spun column. The spun column removes the
digested RNA, is simpler and faster to do than PEG ppt and returns the DNA in
TE in the original volume. Yields material of similar quality too. The spun
columns are home-made and quite inexpensive. I'll be happy to post a more
detailed account if people are interested (it's only
yet-another-plasmid-miniprep). I've been using the miniprep DNA for
restriction digests, sequencing, subcloning and bacterial transformation. I
don't do mammalian cell transfections, so I can't comment on the quality of my
miniprep DNA for that purpose.
One published modification that I have incorporated into the alkaline lysis
part is the increased acetate concentration to precipitate the chromosomal DNA
and RNA after SDS/NaOH treatment. Helge Weissig recently mentioned that
reference (Feliciello and Chinali, Anal. Biochem., 212, 394-401, 1993) in
response to a different question. I think this modification is even more
useful for larger scale (100 ml, liter) preps since it reduces the amount of
RNA in the isopropanol pellet and that makes resuspending the isopropanol
pellet that much easier.
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
email: tan at aeolus.vmsmail.ethz.ch
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