Mysteriously Disappearing DNA

Wolfgang Schechinger hubahopp at gmx.de
Fri Jul 30 04:44:55 EST 2004


Dear Shelley, 

of course it might be some nasty contaminant (the smell of your culture is
ok??), but when it happens repeatedly, it is very likely that the problem
has a more sophisticated cause:

IMHO, the model of dilution which is frequently used for describing the
situation does not reflect the real situation, just the result is the same.
<Darwin>As you have something growing which is ampicillin resistant, it's
obvious that this organism has an advantage over your clone of interest,
can multiply faster and thus simply overgrows it </Darwin>. 

This could mean that 

a) your insert is toxic (inhibits your bug's growth, thus you select for
bugs with an increasingly lower vector number), 

b) there is homologous recombination (your plasmid integrates into the
host's genome. This will frequently happen when your insert is part of the
host's genome, you might find out by PCR) 

c) your amp is bad, in wrong (too low) concentration or you have the wrong
resistance gene / antibiotic (unlikely) or you grow your bugs too long (all
amp already has been hydrolyzed) or your bugs have acquired ampR before by
some reason (contamination / mutation)

Possible remedies / workarounds are 

- grow the bacteria at lower / higher temperature 
- increase antibiotic concentration
- change the strain (see e.g. the list in your NEB catalogue, to be found
somewhere in the tech section)
- use different antibiotic / resistance gene combination (usually means you
have to switch or modify the vector)
- use a low copy vector (or change the ori)
- check the promoter! when it's leaky, you might have some unwanted
expression that puts your bugs simply under stress. XGal inducible vectors
e.g. may be suppressed by including glucose in the broth.

For a more detailed discussion, you'd need to give us details about your
vector, insert and strain. Do you have success with other inserts with the
same system / conditons? This might simplify troubleshooting more than
significantly! You grow more than one prep from your plate, don't you??!!

Best reagrds,

Wolfgang

At 23:23 28.07.2004 +0100, Shelley.Sandiford at lrcc.on.ca wrote:
>
>
>Just a quick question as I am new to cloning.  I have a sample of bacteria
that
>were successfully transformed with my vector/gene of interest.  The
problem that
>I am
>having is that when I attempt to grow a new sample from my positive clone,
the
>vector (along with the gene) appears to be diluted out (i.e. something
>ampicillin
>resistant is growing, but no longer appears to contain the original vector).
>
>Has anyone ever experienced this kind of "dilution" and/or have any
suggestions
>as to what might be happening (contamination, etc)?  I am open to any ideas.
>
>Shelley Sandiford
>
>
>
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>---
>
>
Wolfgang Schechinger


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