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  2. → Viewing Profile: Posts: rrn

rrn

Member Since 27 Sep 2012
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In Topic: Puromycin - shRNA - stable cell line for knock down

06 October 2012 - 04:57 AM

No i did qRT PCR only !! With control cells.. also for gene of interest as well as B-actin for internal control ! p21 was surprisingly highly expressed in the knock down set compared to control cells...

Thats why i was wondering how then these cells survived if they dont have the plasmid inside !

Thanks anyways.. will check up more about the shRNA transfection/functionality !

In Topic: Puromycin - shRNA - stable cell line for knock down

05 October 2012 - 07:32 PM

I did do a puromycin kill curve .. and selected the puromycin amount to be used. Non transfected at same cell density of seeding dies completely..

I did not check at protein level coz at RNA level itself i got p21 PCR product..

If not transfected / construct is bad.. how are these cells surviving with puromycin pressure?

shRNA is in pLKO.1 vector !

In Topic: Puromycin - Stable

04 October 2012 - 12:58 AM

yea thanks ! I removed puro pressure for 2 days and checked for expression of the gene and protein.. its there.. So i guess 24-48 hours without pressure wont affect ..

Thanks again !

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