Growth media

Peter Barrett BARRETT at RASCAL.MED.HARVARD.EDU
Wed Oct 8 22:26:32 EST 1997


To all methods-reagents bionetters,

Just a word of warning.  I recently had the experience that a particular
plasmid of mine, which had worked perfectly fine 6-12 months ago, suddenly
seemed to go kaput on me.  Basically I would inoculate from single colony
directly into either mini (4 ml) or maxi (100 ml) cultures-- bacterial
growth for both was reasonable considering the expected size of plasmid.=20
Nevertheless, the mini cultures showed seemingly no plasmid at all (or a
smear where there should have been plasmid), the maxi cultures had either
a very weak plasmid band of a larger-than-expected size, or else a very
huge amount of a much smaller size plasmid.  Neither of these turned out
to be the right thing as checked by restriction digest.  (In other words,
I was getting complete junk out of this; going back to my glycerol stocks
did me no good, nor did retransforming what little original plasmid I had
left-- I got the same thing again in each case.  I was looking at either
troubleshooting it through or recloning-- not a welcome prospect,
considering that it wasn't the most straightforward of clonings.)

After quite a bit of troubleshooting, including changing antibiotics,
growth conditions, method of preparation, etc., I discovered that the
problem was, voil=E0, the media.  At one point we switched over from making
our own to a commercial source which comes in the "capsule" form.=20
Inoculating from single colonies into either commercial superbroth from
capsules versus homemade LB made *ALL* the difference-- this plasmid would
*not* grow properly in commercial superbroth-- apparently seeming to
recombine-- but grew beautifully in homemade LB (but note that we have
other plasmids that seem to grow fine in this commercial superbroth).  I
also know that this plasmid/bugs grow fine in homemade superbroth because
that is what I had been using before when I had grown it originally.  All
I can think is that somehow the difference in broth put some sort of
negative selective pressure on the bugs/plasmid???=20

Some other relevant details: the clone is an 18kb pUC18 derivative with
lots of added sequences, carries AmpR marker.  The bugs were TOP10F',
which have TetR on the F'.  Both antibiotics were used for growth.=20
Purifications were boiling for minis or Qiagen for maxis.=20

Caveat emptor!=20

Best,



Peter=20
Peter Barrett=20
Graduate Student=20
Harvard Medical School and Harvard University

Mailing Address:
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Children's Hospital
320 Longwood Avenue
Boston MA  02115
Tel: (617) 355-7586
Fax: (617) 730-0422
email: barrett at a1.tch.harvard.edu
               ^^ letter "A", number one




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