No colonies- toxic or unligated?

EK someone at microsoft.net
Wed Jan 28 12:48:54 EST 2004


I think if you do your cloning is a bit larger scale, you would be able to
see the product on a gel (you might have to use fluorescence detection with
SYBR gree instead of EtBr though). Also, you could do resctiction analysis
of the plasmid with the insert to prove that the cloning was successful. You
could also recut and religate you plasmid with the insert so that the insert
is removed, and see if that increases the number of transformants compared
to the noncut plasmid with the insert.
-Emir

"Mélanie Tremblay" <mel_4_7 at yahoo.ca> wrote in message
news:4017147D.2070704 at yahoo.ca...
> Hi
>
> I amplified my gene (2.3kb) with primers containing restriction site
> (and 6 extra bases), after I digested with NcoI and XmaI and ligated in
> vector (digested and dephosphorylated). Few colonies (about 10) were
> produced in XL-10 and no one positive with control positive ok.
> After that, I tried TOPO TA cloning. I followed protocol and obtained
> few colonies (around 10) with no one containing insert (with TOPO10F').
>
> My insert is probably toxic, but how I can know if it's the cloning that
> failed or it's the bacteria that not support it? This gene is presently
> encoded in pbluescript in XL-10 cells. Growth is slow but I can produce
> a little bit of DNA.
>
>
> I'm desperated!!! Can you help me?
> Mélanie Tremblay
>





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