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Dual Luciferase Assay


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#1 Sank

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Posted 13 January 2010 - 09:04 AM

I am planning to do a dual luciferase assay using the firefly and renilla luciferase vectors.
Can someone suggest me a ratio for the co-reporter vectors to be added to the transfection mix.

Regards
Sankella

#2 StevieRay

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Posted 13 January 2010 - 09:13 AM

Your best bet is to first test them at different ratios, say ranging from 1 luc: 1 ren to 1000 luc: 1 ren. The reason is that luciferase and renilla constructs can have different activity, depending on the cell line you transfect them into. Renilla will vary some (up to 10-50 fold depending on the cell line and the promoter it is driven by), while luc can vary considerably, depending on your promoters activity in the cell line you are testing.

#3 Sank

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Posted 13 January 2010 - 09:21 AM

Thank you Steve that helps :rolleyes:

#4 KevinK

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Posted 14 January 2010 - 12:23 PM

Sankella,
In addition to the good advice StevieRay has provided, it is also important to make sure that the presence of the Renilla control is not altering the expression of the firefly (usually the experimental) vector. In general you just need enough Renilla expression to be consistenly above background. The Dual-Luciferase Assay chemistry will quench the firefly signal 10,000-fold, so this more has to do with maintaining more normal biology than any concenr over the signals. I think you may find an article I co-wrote useful: http://www.promega.c...17/cn017_09.htm. See figure 3 especially. I hope this helps answer your question. Also, please always feel free to contact Promega Techncal Services by phone, e-mail, or chat.

Regards,
Kevin
Promega Corporation
Madison, WI




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