superbroth and plasmid preps

afc at gnv.ifas.ufl.edu afc at gnv.ifas.ufl.edu
Wed Aug 26 15:14:56 EST 1992


Last week I posted a question about the use of rich broths for 
growing plasmids.  Based on the replies that I received, it seems
that most people who have tried them like them.

The key elements seem to be 1) more nutrients, 2) better buffering, 
and 3) more aeration.

Some editted responses are included below.  I thank the responders
for recipes and comments.

Andrew Cockburn
USDA


From: READY at UTBC01.CM.UTEXAS.EDU
  
      	For plasmid preps (both large scale and mini) I habitually use 
2xYT + 0.2% glucose. 2x YT is (per liter):  
16 g Difco Tryptone
10 g Difco Yeast Extract
5 g NaCl

I confess I have never quantitated the difference in yield between this and
standard broth, but eyeball measurement has indicated it to be significant.
Typical yields for 10 ml minipreps are 50-100 ug for pUC-like plasmids. My best
1 liter prep was > 9 mg plasmid. We get different yields from different strains,
but in general we get better yields from "upscale" media. Whether it's worth
it is up to you and probably depends on what your plasmid copy number is.
Most of us would not use up 9 mg of plasmid in a century, so it may be overkill.
(However, we use pUC for restriction and transformation controls all the time -
my 9 mg only lasted about 2 years.)  

Good luck,
Mike R. 

From: Jeffrey N Masters <jmasters at magnus.acs.ohio-state.edu>

I personally like the rich media and use it routinely for plasmid
preps My lab uses terrific broth (detailed in Sambrook et al. 
((the new Maniatis manual))). The advantages are detailed in the
latest Focus from BRL (vol 14, #3, in an add after page 94) while
the original is in vol8, #2 pg 9. A 10 ml miniprep lasts a long time.

From:	IN%"wjb1 at midway.uchicago.edu"  "William J. Buikema"

	I grow all of my E. coli strains in a special rich medium that I 
call LB-M9.  I'm sure there are other variants of it around, but this is
my special version.  It contains standard LB (1x, although higher concs. can
be used), and slightly modified minimal medium M9 salts.  The M9 salts 
provide NH4+ and phosphate buffering, while the LB supports rapid growth.
The carbon sources that I use are a combination of glucose and Na succinate.
I found that in straight glucose medium E. coli tends to rapidly acidify and
self-limit its growth.  In succinate the opposite pH change occurs.  With a
mixture of the two, the pH is very stable around 7.2 and the culture attains
a very high density and maintains its health for much longer during long
culturing (i.e. if LB cultures hit stationary phase too early, one gets much
more chromosomal DNA in a plasmid prep, presumably due to cell breakdown).
O2 limitation becomes more of an issue, and the highest densities can only 
be reached with vigorous aeration. 
	I can send you the exact recipe that I use if you are interested.

From:	IN%"BMA14432 at VAX1.UTULSA.EDU"

The literature your looking for was in a publication called Focus. The results w
ere the same as they previously published/issued:
Media called TB(Terrific Broth) works best.
This is what we use routinely in our lab.
Typically from a liter Culter we get 2-5 mg or plasmid(4-7 Kb).
Don't have the recipe at hand but if no one else gives it to you
feel free to e-mail me for it.
Mark Brudnak

From: minagawa at nemo.life.uiuc.edu (Jun Minagawa)

Now, rich media is very popular. I have no problem of any vector and host.
The yields are excellent (about 2-10 times of LB).  A typical recipe is
on Molecular Cloning Ed.2 as a Terrific Broth. You should try it !

From: MIC268A at vaxc.cc.monash.edu.au

I have found the following media to be extremely good for high plasmid yields.
The reference for this media is Focus 9:2 page 12. It is by Tartof and Hobbs
(1987), "Improved media for growing plasmid and cosmid clones"

The authors claim that the media gives from a fourfold to a tenfold increase
in the yield of DNA per 100ml of culture. This was compared to LB, LB+glucose
and the plasmids used to compare yields were pUC18, pUC18+13kb insert and a
cosmid with a 35kb insert

In my hands I have found this media to be beneficial when attempting to get
high yields of low copy number plasmids (ie. pSC101 derivatives) and as for
higher copy number plasmids (ie. pUC18) I've ended up with DNA to last a few
years.

From:	IN%"bex at acpub.duke.edu"

	     I have made many large preps of plasmids that are close to 30
kb in size.  I get very little DNA when using LB broth 1X but a good amt
when using 2X.  I don't have exact comparisons but usually 2X broth gives at
least 2X the DNA.  I use primarily pBS vector in DH alpha1 host cells.

We use the LB media from Maniatis

10 g Bacto tryptone
5 g Bacto yeast
10 g NaCl  
ph 7.5

For 2X broth double tryptone and yeast but not salt



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