Please help with stable transfection

Wolfgang Schechinger hubahopp at gmx.de
Fri Oct 4 04:43:02 EST 2002


Khoa, 

I suggest to try a different analytical method (e.g. PCR, western or
southern lot). 

Woe


At 03:31 03.10.2002 +0100, Khoa Ngo wrote:
>Hi 
>  I am in the middle of the preparation of a stable transfectant clone
>with RAW264.7 murine macrophage cell line. For which, a source of about
>50% positive stable transfectant cells was used for a limiting dilution
>with a ratio of 1/3 cell per well in a 96 well plate. Non-transfectant
>cells, used as supporter cells, were added together with the
>transfectant cells to the wells in medium without G418. After a few
>days, G418 was added to kill the supporter cells and in about 10 days,
>colonies were clearly seen under microscope for about 30 wells. From
>these, only 13 wells had colonies visuable to the naked human eyes, thus
>cells from each of the 13 wells were screened for positive clones.
>However, fluorescent microscopic observation (after immunofluorescence)
>shows some head-aching results for me. It is supposed to show either non
>or all cells are positive, but non of the clones could demonstrate a
>complete batch of positive cells, most of the clones have a few positive
>cells, while some show approx. 30 - 40 %  positive cells, which is less
>than the original source (a sample of the original source was screened
>to confirm this lesser effect).
>
>     Here, i am wondering if someone can tell me what could have
>happened in order to lead to such results? and what would be the posible
>options available to get the expected clones? 
>
> I am really greatful for your response.
> Many thanks in advance
> Kind reagards
> Khoa
>
>     
>
>
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