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Stable Cell Line generation


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#1 Winged Monkey

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Posted 22 June 2011 - 09:18 AM

Hi, I am currently working to generate several stable cell lines using HEK 293 and HeLa cells. Most of my plasmids are pcDNA3 and I am able to use G418. The HEK 293 seems to work, but my HeLa's don't express the plasmid when I use them in my uptakes. I use TransIT LT-1 for my transient transfection and allow the media to remain on the cells 24-48 hours before removing and adding selection. Suggestions???

I also have pEGFP plasmid that I know very little about. It was Kanamycin resistant when I prepped the DNA. What should I use to try to select for it after transfection?

One more dilemma, do you usually do a kill curve for puromycin or is there a standard amount you should use? I have plasmid with the pKH vector that uses it.

Do you have a site that is a good reference place for someone with a degree in chemistry trying to work very diligently in cell biology???

Thank you!!!

#2 bob1

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Posted 22 June 2011 - 07:55 PM

Always do a titration of the quantity of drug needed to effectively kill the cells before transfection. Check the product manuals for appropriate ranges of concentration.

Do you have a method to check that your transfection is working? If so - use this to test appropriate DNA:transfection reagent conditions for the HeLa.

The pEGFP plasmids I know don't have any eukaryotic selection markers on them.

#3 leelee

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Posted 22 June 2011 - 08:14 PM

Your pEGFP vector, is it pEGFP-C1 by any chance? The KanR marker is also a neomycin resistance marker (G418). In fact I think many of the pEGFP vectors that have Kn resistance use the same gene.

Edited by leelee, 22 June 2011 - 08:16 PM.


#4 Winged Monkey

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Posted 23 June 2011 - 07:30 AM

Thank you both. I will do my curve for puromycin as well.




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