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rrn

Member Since 27 Sep 2012
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Cellular Senescence and Signaling

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drwho
04 Oct 2012 - 11:12
2xzwei
28 Sep 2012 - 01:18

Topics I've Started

Puromycin - shRNA - stable cell line for knock down

05 October 2012 - 05:50 AM

Hi,

I have this sh RNA construct against p21 which has puro resistance. After transfection and pressure.. very few cells.. less than 100 only would have survived.. and they started dividing rapidly and now with the same amount of puro.. there is no cell death. But when I isolated mRNA and made cDNA and did a real time for p21.. the transcript is still there. Should I design different sets of real time primers and check ?

If the stable selection did not work then why the cells are not dying now ? Can cell lines develop puro resistance by any chance ?

Thanks,

RRN

Puromycin - Stable

27 September 2012 - 08:09 PM

I have generated stables with puromycin res construct which contains a gene coding for a chemokine of my interest. I need to use the spent media to culture other cells in it ! I guess the puromycin contained in the spent media is not allowing the new cells to grow. Is there any way to remove puromycin ? Is it ok if I do not add puromycin for few days and use that spent media ? Will the stable expression get lost ?

Thanks