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Luciferase Assay Help!!


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4 replies to this topic

#1 jasonwu

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Posted 20 March 2005 - 09:19 PM

I transfected DNA-luciferase reporter gene construct into cells and treated cells with some drug, after 24 hr, i measured the luciferase assay through luminator. But i got some readings as zero :( . What is wrong with it?? Does anyone have this kind of experience?? thx.

#2 zienpiggie

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Posted 20 March 2005 - 09:47 PM

How's your control's reading?

#3 thomasleon

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Posted 20 March 2005 - 09:48 PM

Which kit/reagents are you using?
I use the Dual-Lucfierase Kit from Promega, it's pretty good actually. Anywayz, I still got reading even it's the blank sample. Would it be any careless mistake, for example, the machine is not primed? :(

#4 jasonwu

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Posted 20 March 2005 - 10:24 PM

I also used dual-luciferase kit from promega, i cotransfected my DNA and TK(as control), the reading for my DNA is zero but it is non-zero for TK. What do u mean by control, is it those samples without drug treatment?? they also have zero readings.

And what do u mean by primed machine?? thanks.

#5 JunieMoon

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Posted 22 March 2005 - 04:22 PM

I also used dual-luciferase kit from promega, i cotransfected my DNA and TK(as control), the reading for my DNA is zero but it is non-zero for TK. What do u mean by control, is it those samples without drug treatment?? they also have zero readings.

And what do u mean by primed machine?? thanks.

<{POST_SNAPBACK}>


HI,
I've done a kazillion luciferase assays. These are a few of the things that I can think of:

If you are using the luminometer correctly, then "0" means it didn't work.

It could be a bad transfection, bad kit reagents (although unlikely, they are pretty robust), if you are using auto-injectors...is there reagent in them? (priming).

Did you blank it with reagent (no lysate added) (or at least checked what the background might be)?

is it accidentally/automatically doing something you don't know about?

Has somebody else used it recently to confirm that the luminometer is working ok?

It sounds like you have an ok control (TK transfected), but have you also tried non-transfected? If TK is showing some type of reading, could you have mixed up the samples?

Are you sure that your construct works they way that you think it does?

A few times when I've made construct with a tissue specific promoter, it didn't respond as well as I'd hoped, and my reading were near background.

Hope this helps!
Junie




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