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Extra plasmids in minipreps?

Extra plasmids in minipreps? - Protocols and Methods Forum

Extra plasmids in minipreps? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-21-2003, 11:16 AM
Tarja Kokkola
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Default Extra plasmids in minipreps?



Hi everyone!

We have recently encountered problems with our plasmid DNA minipreps. We ran
out of the particular DNA batch used for transfections and started making
more DNA by transformation of our home-made competent DH5alpha E.coli cells,
plating and starting overnight miniprep cultures from single colonies. The
DNA used for transformations has been sequenced, is clean and has worked
well in transfections. No extra bands are visible in this prep.

Problem:
On agarose gel with undigested plasmid prep, we see our plasmid of ~ 6.7 kb
(pcDNA3 plus our gene in EcoRI/XhoI cloning sites) plus faint bands of ~ 8
kb and 10 kb. The extra bands (plasmids?) are digested by restriction
enzymes with the correct plasmid to produce some more faint extra bands. All
our DNAs have previously always been free from these extra bands.

We have checked the following things:
-picked several well-isolated colonies; the amount of 'extra' DNA varies
from colony to colony but is visible in each of them
-transfected competent cells using another DNA preparation but the 'extra'
bands are still visible
-bought a new miniprep kit; nothing helps

Do you have any suggestions how to overcome this problem and obtain clean
DNA for transfections? Your help will be very much appreciated,

Tarja Kokkola



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  #2  
Old 08-21-2003, 11:37 AM
Nina Baltes
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Default Extra plasmids in minipreps?

In article <bi29hj$74d8$[Only registered users see links. ].fi>, [Only registered users see links. ] says...


If your DNA used for the transformation is clean, and you see these
extra bands when using different DNA, it sounds like your competent
cells are the problem. I'd obtain a new batch of DH5alpha, or at least
make new competent cells form the lab stock and check them (i.e. do a
miniprep and check for the bands, there should be none) before
transformation. Or use a different strain.

Nina
--
C'est les microbes qui auront le dernier mot.
Louis Pasteur
[Only registered users see links. ]
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  #3  
Old 08-21-2003, 02:26 PM
EK
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Default Extra plasmids in minipreps?

Are you sure that you are not looking at bands corresponding to supercoiled
state of your plasmid? It is very usual to see extra couple of faint
(sometimes not so faint) bands when running a plasmid on an agarose gel. Try
briefly boiling your samples and see if extra bands disappear. Also, streak
your transformed bacterial strains out on plates with other antibiotic than
required to maintain the plasmid, and see if cells are surviving, which
would be an indication of contaminating plasmids. I have a feeling that you
don't have a problem there, and just misinterpreting your electrophoresis
results.
Emir

"Tarja Kokkola" <[Only registered users see links. ]> wrote in message
news:bi29hj$74d8$[Only registered users see links. ].fi...
ran
cells,
kb
All


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  #4  
Old 08-22-2003, 05:47 PM
Jens Mollerup
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Default Extra plasmids in minipreps?


"Tarja Kokkola" <[Only registered users see links. ]> wrote in message
news:bi29hj$74d8$[Only registered users see links. ].fi...
ran
cells,
kb
All

It might be single stranded DNA. Try to run an agarose gel without
ethidiumbromide and stain afterwards. Single stranded DNA should run
differently in the presence/absence of ethidiumbromide in the gel.

Jens


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  #5  
Old 08-28-2003, 06:47 AM
Tarja
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Default Extra plasmids in minipreps?

> > We have recently encountered problems with our plasmid DNA minipreps. We


Thank you for your replies! Single stranded DNA or the supercoiled
state of the plasmid (suggested in the previous reply) cannot explain
the extra bands we see after restriction digestions. SS DNA would not
be digested with restriction enzymes, I suppose. Different enzymes
give different restriction patterns also for this 'extra' DNA. Some
extra fragments are smaller than any of the expected fragments with
EcoRI, for example, indicating that the extra bands cannot be a result
from uncomplete digestions.

We will try to use different competent cells next time. If you have
any other suggestions, please send them to this newsgroup!

-Tarja
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