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Plasmid prep issues

Plasmid prep issues - Protocols and Methods Forum

Plasmid prep issues - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 04-07-2007, 02:04 AM
peter
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Default Plasmid prep issues



Hi group,
I have some bad bacteria recently that won't give me any plasmid on a
miniprep. I tried almost everything and it gets more and more bizarre.
First I tried Qiagen - and I have some controls that I know prep well.
all of them have AmpR, all were grown at the same time, media,
incubator. As a result, the controls had ton of plasmid, and the
bizarre ones had none. Then I plated again on an agar - all grew, the
ones that don't give plasmid were still resistant, with a slightly
bigger colonies than the other. I start questioning the Qiagen kit - I
took some of my cultures and just boiled at 95oC for couple of min.
and then loaded on gel - same result, the ones that always have
plasmid still have it. So I went and I run them on FACS to see if the
"bad guys" are not yeasts - they are not, then I looked under
microscope - they look like each other , but the ones that fail to
give plasmid are much more motile , compared to the ones that give me
plasmid. At that point I gave up and wrote this asking for your
wisdom.... any ideas why these E. coli won't have a plasmid, but will
have AmpR?
Thanks,
Peter
P.S. these are all similar size plasmids

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  #2  
Old 04-07-2007, 02:06 PM
WS
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Default Plasmid prep issues

Hi Peter,

Don't blame it on the kit, seems to me rather that your bugs have
integrated the plasmid into their genome. Are you using a recombinase
minus strain? Is your insert sort of bacterial gene that would permit
homologous recombination?

Wo

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  #3  
Old 04-07-2007, 05:01 PM
Pow Joshi
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Default Plasmid prep issues

On 6 Apr 2007 19:04:31 -0700, peter <[Only registered users see links. ]> wrote:



my understanding is that ampicillin plating tends to give you suppressor
colonies after a while, surrounding the plasmid containing clone, when the
ampicillin is used up ...... it is a good idea to incubate the agar plate
for max 12 hrs, maybe less ..... you seem to have lost the plasmid somewhere
along the line ..... if you still have the original plasmid, probably you
could try transforming again....

that's a parsimonious explanation i can come up with.
hope it helps
pow


P.S. these are all similar size plasmids
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  #4  
Old 04-07-2007, 10:50 PM
peter
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Default Plasmid prep issues

On Apr 7, 10:06 am, "WS" <[Only registered users see links. ]> wrote:

Integration is the only meaningful explanation, although this happens
across many different strains, none of which is rec positive. It seems
that somehow becteria integrated the plasmid in the F' or regained
intact F episome . Inserts also are different and some of the codon
optimized ...

Peter

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  #5  
Old 04-07-2007, 10:53 PM
peter
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Default Plasmid prep issues

On Apr 7, 1:01 pm, "Pow Joshi" <[Only registered users see links. ]> wrote:



I plated same bacteria that did not give plasmids, I did not make new
transformation, so I don't have satellite problem.

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