Plasmid DNA spont. digesting - the long version :-)

Sean Yoder syoder at chuma.usf.edu
Sun Jun 6 09:55:19 EST 1999


     Wow this goes back a few weeks....I concluded that there was
contamination of some sort.  Every stock that a certain undergraduate
touched ended up becoming contaminated.  He thought that if he wore
(nonsterile) gloves he did not need to use aseptic technique...
     So I transformed new cells just to find that the insert was never
there to begin with...looking through the undergrad's notes I now see
that he never screened properly....so I started it over subcloning into a
more appropriate vector for my project....
    I have learned a lot in the past couple of months....and one lesson
is not to take over another person's work ....just do it yourself....

Sean Yoder
Microbiology Graduate Student
University of South Florida

plxsrt at pln1.nott.ac.uk wrote:

> If I am reading your essay correctly you get growth of the host cells
> without plasmid under Amp selection.  This is not what you expect-
> your cells are contaminated, get a fresh stock and start again.
> Everything else you describe could arise from contamination.
>
> If you stil get extra bands after the maxiprep and after you've ruled
> out the strain it may be from RNA contamination (have you added RNase
> to the cell resuspension buffer(1)?) or it may be due to poor timing
> of prep stages  in which case you can get single stranded DNA coming
> through.
>
> Simon T




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