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transfecting Jurkat with Fugene HD: does not work?

Fugene HD luciferase vector jurkat

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4 replies to this topic

#1 et2b

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Posted 25 August 2013 - 10:57 PM

Hi all,

 

We are busy doing luciferase assays (dual). We would like to do a transient transfection of Jurkat.

Since Promega has a protocol in its database we bought Fugene HD as transfection agent. 

 

It works extremely well on HepG2, but we have absolute zero success with Jurkat.

 

We tried 1.5:1 -8:1 ratios in +1 steps, tried 3x105 and 5x105 cells, and both 24h incubation and 36h incubation.

We are using IMDM 2mM L-glut, 10%FBS and pen/strep as culture medium.

 

Does anybody have good tips/protocols to share how to transfect Jurkat with a luciferase vector?

Preferably with Fugene HD, but at this stage we'll settle for anything that works...

 

We are stuck...

 

best

 



#2 Curtis

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Posted 27 August 2013 - 12:44 AM

Different cells need different protocols. I am not sure about Jurkat, but we have this hamster cell line called BSR T7/5 in the lab and the person who made them only recommends calcium-phosphate transfection. He says lipo or Fugene transfection won't be effective. Also, last time I transfected CAMA-1 and MCF7 with luciferase vector, and it worked better with calcium-phosphate. Personally, I still prefer calcium-phosphate to any other reagent. 



#3 et2b

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Posted 27 August 2013 - 01:14 AM

Hi Curtis, many thanks for your reply. Do you have a protocol for the calcium phosphate with mammalian cell lines? I only knew of this option in combination with bacteria.

Many thanks



#4 Curtis

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Posted 28 August 2013 - 01:12 AM

I do have, but need to find it in my thesis. You can check Youtube or find better protocols at Invitrogen website. Sigma also has a kit for it. Maybe you can check the manual.



#5 et2b

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Posted 28 August 2013 - 01:22 AM

RFTM, yes I have done so. Contact with promega the supplyer, I have.

Its not solving my Jurkat problems.

best wishes







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