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HeLa tranfection- Dual luciferase Assay


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#1 Lulus

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Posted 28 June 2009 - 12:12 PM

Hello,
So I have a few questions. I have been trying to do co-transfection of a firefly construct and a renilla plasmis, to be use as background, in HeLa cells. I'm currently using Lipofectamine LTX from Invitrogen. When I called Invitrogen they suggest me to use that transfection reagent. The protocol that I'm usins is the one suggested by Invitrogen as well. Here is the liknkhttp://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/protocols.Par.89049.File.dat/Human_cervical_carcinoma.pdf . I'm usins HeLa cells no HeLa S3 cells. I grow the cells in a 96 well plate until they are 80% confluent and then I follow the Lipofectamine LTX for HeLa H3 protocol. After 24 hs my cells look good. I use a GFP construct as a control for transfection. After 24hs I want to read transcription of my transfected plasmids usin the Dual luciferasa Assay Kit from promega. The readings that I get are low. The firefly in a 96 well plate reads between 8 to 100 and the Renila goes from 25 to 250. I don't think this is an aceptable reading but I don't know what to do to get better readings.
I'm transfectin 50ng of DNA and 0.175ul of lipofectamine. The GFP control looks OK before I lyse the cell to do the assay.
Has anyone had the same problem? Should I use Lipofectamine 2000? Should I wait 48 hs before I do the luciferase assay?

Hopefully someone can help me out here. Thank you!

#2 jah

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Posted 29 June 2009 - 05:10 AM

I have two suggestions.
1) Wait a little longer before testing Luciferase expression
2) Try an optimization experiment, trying different ratios of DNA to lipofectamine, and different ratios of Firefly to Renilla (I use 1:6).

Also, are you using clear-well plates? If so, you might want to try opaque walled plates which reflect the luminescence produced within each well, rather than let it 'leak' into adjacent wells. Good Luck

#3 genehunter

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Posted 29 June 2009 - 09:10 AM

Hello,
So I have a few questions. I have been trying to do co-transfection of a firefly construct and a renilla plasmis, to be use as background, in HeLa cells. I'm currently using Lipofectamine LTX from Invitrogen. When I called Invitrogen they suggest me to use that transfection reagent. The protocol that I'm usins is the one suggested by Invitrogen as well. Here is the liknkhttp://www.invitrogen.com/etc/medialib/en/filelibrary/pdf/protocols.Par.89049.File.dat/Human_cervical_carcinoma.pdf . I'm usins HeLa cells no HeLa S3 cells. I grow the cells in a 96 well plate until they are 80% confluent and then I follow the Lipofectamine LTX for HeLa H3 protocol. After 24 hs my cells look good. I use a GFP construct as a control for transfection. After 24hs I want to read transcription of my transfected plasmids usin the Dual luciferasa Assay Kit from promega. The readings that I get are low. The firefly in a 96 well plate reads between 8 to 100 and the Renila goes from 25 to 250. I don't think this is an aceptable reading but I don't know what to do to get better readings.
I'm transfectin 50ng of DNA and 0.175ul of lipofectamine. The GFP control looks OK before I lyse the cell to do the assay.
Has anyone had the same problem? Should I use Lipofectamine 2000? Should I wait 48 hs before I do the luciferase assay?

Hopefully someone can help me out here. Thank you!

If you increase the reporter dosages to at least 0.1, maybe 0.2 ug and give another try first, let us know if this helps.




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