(no subject)

[Michael Sullivan]

If your culture is indeed "mixed" it would almost certainly NOT be at  
the level of having two different plasmids in a single bacteria. So  
the only likely possibility for a mixed culture would be one  
containing two different populations of bacteria, some of which have  
one plasmid, some of which have the other plasmid. To separate these,  
you would only need to streak out some of the culture for single  
colonies on a selective plate, pick several, and characterize the  
inserts (either by restriction digestion or by sequencing). If this  
is what your problem is, then by reisolating single colonies, you  
should be able to identify the clone you are looking for.

Can you tell anything about the nature of the sequence that is  
superimposed? I'm assuming that you are just sequencing to confirm  
everything is OK in your clone before taking the time to transform  
plants (?), and that you know what sequence to expect. If that is the  
case, you might be able to tell what the superimposed sequence is,  
and that might give you a clue as to what is going on.

Hope this helps.

Mike

On Mar 28, 2007, at 12:36 PM, Orsini Elena wrote:

> Hi,
>
> I have a problem in my sequencing results. I cloned a GUSNOS insert  
> into
> a plasmid and afterwards a promoter in front. When I received my
> sequencing results the forward sequence of my promoter was fine but  
> the
> reverse ( the annealing was at the beginning of the GUS gene) was
> superimposed after 130 base pares. This happened exactly at the  
> level of
> the cloning site before the GUS fusion.....
>
>
>
> I suspect that my culture contains a mixture of two plasmids, the
> transformed and the not transformed....or in the worst case two
> different repetitive sequence of the GUSNOS.
>
> How could I figure this out?
>
> Is it possible to separate eventually the two plasmid?
>
> Thank you very much
>
> elena
>
>
>
>
>
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)