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stable cell line


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#1 SF_HK

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Posted 23 December 2009 - 01:16 AM

HI,

I transfecetd my cel lines with gene of interest in a 6 well plate. After 24 hrs, I had split the cells in 6 well plate to 2 10cm dishes and 48hrs after transfection I started added 500ng/ml g418. Its been 2 weeks under selection and my plates are conflent. I think I added too many cell sper dish. Would it be wise to split my cells ithat are in the 10cm dish to another dish with fewer cells? My cell line has a good transfection efficiency but I may have made a mistake my plating too many cells. Its been two weeks and no colonies are formed since the plate is to confluent. Since no colonies have formed is it wrong to assume that the cells may have lost the expression of my gene, even though they were under selectiona all this time for 2 weeks?

#2 jah

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Posted 23 December 2009 - 09:15 AM

The timing of your splitting is exactly what I do. However I wonder if your dose of G418 (50nanog/ml) correct? It seems very low. If you haven't yet, I'd recommend doing a kill curve with non-transfected cells from 500microg/ml-10ug/ml. In my experience, most tumor and primary cells are resistant to doses less than 100ug/ml.

#3 SF_HK

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Posted 28 December 2009 - 11:06 PM

The timing of your splitting is exactly what I do. However I wonder if your dose of G418 (50nanog/ml) correct? It seems very low. If you haven't yet, I'd recommend doing a kill curve with non-transfected cells from 500microg/ml-10ug/ml. In my experience, most tumor and primary cells are resistant to doses less than 100ug/ml.


HI,

Sorry there was a typo. I'm using 500ug/ml of G816. Do you think it was wise to split from the 10cm culture dish?




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