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Problem with stable clone


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#1 jaya2020

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Posted 31 December 2014 - 07:18 PM

Hi,

 

Iam facing a problem with stable clone.I think Iam having a problem but not so sure. I have c3h10 cells which is a stable clone with G418 resistance and the stably expressing protein is a cell cycle probe for G1 (red tag). In this I have to insert a protein (green tag) which is localized to the nucleus, which has puromycin resistance. I followed the standard procedures,selected using G418 and puromycin, and i looked for the green and red fluorescence. What I saw was that, not all cells were expressing both the tags. There were some cells which didnt express both.

So, I assumed that the cells not expressing both the tags may be in the G2 /S or M phase. is this correct or is something really wrong with my expt. please help.

Jaya



#2 GNANA

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Posted 01 January 2015 - 09:35 AM

I am not an expert in cell cycle stuffs, but looking superficially i am getting some thoughts, the cells you working are mass culture or isogenic clones? if mass culture the no expressing cells could be due to no or low level expression of your probe.  

 

secondly did you synchronize cells and followed your probe?


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#3 jaya2020

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Posted 02 January 2015 - 10:49 AM

It is isogenic clones. I did nt do any cell sync as Im screening the colonies for the desired expression.






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