weird results after transformation

Sometimes, I've gotten the insert somewhere into a different region of the
genome. Have you checked by PCR whether you do have the Kan resistance gene?
It might be that the flanking sequences you have are not long enough or have
also homology to another DNA region, and you are getting them integrated
there.

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Today's Topics:

1. weird results after transformation
(Margarida Isabel Rosa Bento Palma)


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Message: 1
Date: Tue, 3 Apr 2007 17:25:52 +0100 (WEST)
From: "Margarida Isabel Rosa Bento Palma" <[Only registered users see links. ].pt>
Subject: [Yeast] weird results after transformation
To: [Only registered users see links. ]
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<45056.193.136.124.168.1175617552.squirrel@webmail .fct.unl.pt>
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Dear all

Last time I wrote I was having problems with my yeast transformations. The
ones that answered me gave me good advices (I didn't know that I should
plate first in YPD and after replica plate in YPD+G-418) and i finally got
some colonies. Now I am having other problems. I am doing the insertion of
a fragment into S. cerevisiae chromossome (polyploid strain) by homologous
recombination. The insertion of my fragment will give this strain the
capacity of grow in G-418 because it contains the Kanamicin gene. I got
some S. cerevisiae colonies resistant to G-418 but apparently they don't
contain the insert (I have tested by PCR and Southern). Have you noticed
some similar results? Isn't weird that the strain grows without the
insert? I am sure that my initial strain doesn't grow in G-418.

Thank you

Kind regards,

Margarida



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