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Transfection Issue


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#1 moorele

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Posted 14 July 2009 - 06:16 AM

Hi,

I have a pcDNA3.1 vector which has my gene of interest cloned in with a FLAG tag so total vector size ~8.5kb. I tried transfecting MCF-7 and MDA-MB-231 cells with this vector - both lipofectamine 2000 and nucleofection (amaxa) but when I probe western blot I'm not getting any bands. Positive control working so i know antibody isn't the issue. I then transfected HeLa cells with the same vector & it worked fine - got right size bands on western blot.

I'm going to nucleofect the two cell lines with a control GFP vector to see if transfection is the issue. Are these cell lines generally hard to transfect? They are used so widely I would have thought they shouldn't be too difficult.

Any idea's as to why I cant get this vector into these cell types?
Many thanks!
moorele

#2 genehunter

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Posted 14 July 2009 - 08:03 AM

I remember I had about 50% EGFP positive cells for both lines with lipo2000. They are responably easy to be transfected.

#3 moorele

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Posted 14 July 2009 - 08:16 AM

I remember I had about 50% EGFP positive cells for both lines with lipo2000. They are responably easy to be transfected.



Ok cool... thats good to know! Hmmm I wonder what i'm doing wrong! I'll nucelofect in pMax-GFP over the next few days & see how I get on!




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