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Help with cloning


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6 replies to this topic

#1 Penguin

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Posted 19 June 2009 - 08:39 AM

Hi,

I want to generate a plasmid for mammalian expression in which my reporter gene is downstream of an E2F1 promoter sequence. The standard mammalian expression vectors have multiple cloning sites downstream of CMV promoters, to ensure that I only see expression of my gene upon E2F1 transactivation do I need to remove the CMV promoter??
Or, does anyone know of any vectors which have no promoter sites that I could use instead??

Thanks!
P

#2 cellcounter

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Posted 19 June 2009 - 11:39 AM

Hi,

I want to generate a plasmid for mammalian expression in which my reporter gene is downstream of an E2F1 promoter sequence. The standard mammalian expression vectors have multiple cloning sites downstream of CMV promoters, to ensure that I only see expression of my gene upon E2F1 transactivation do I need to remove the CMV promoter??
Or, does anyone know of any vectors which have no promoter sites that I could use instead??

Thanks!
P


You certainly need to remove the CMV promoter.

#3 liweixie

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Posted 21 June 2009 - 09:41 AM

Hi,

I want to generate a plasmid for mammalian expression in which my reporter gene is downstream of an E2F1 promoter sequence. The standard mammalian expression vectors have multiple cloning sites downstream of CMV promoters, to ensure that I only see expression of my gene upon E2F1 transactivation do I need to remove the CMV promoter??
Or, does anyone know of any vectors which have no promoter sites that I could use instead??

Thanks!
P


I also work on promoter related project.
u can use promega company PGL3/ 4 vector, that is promoter less. u can insert your promoter region into the mutil cloning region just in front of the reproter gen luciferase.
transfect the construct vector into the mammalian cells with any transfect method. and detect the luciferase with promega company's dual luciferase kit.

#4 Penguin

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Posted 21 June 2009 - 02:22 PM

I also work on promoter related project.
u can use promega company PGL3/ 4 vector, that is promoter less. u can insert your promoter region into the mutil cloning region just in front of the reproter gen luciferase.
transfect the construct vector into the mammalian cells with any transfect method. and detect the luciferase with promega company's dual luciferase kit.


Yeah we have these constructs in the lab but I wanted to use a destabilised GFP I've just got hold of so I can do FACS analysis. Do you think I could replace the luciferase gene with my GFP??

P

#5 Rsm

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Posted 22 June 2009 - 05:38 PM

If you have the original pTAL-d2EGFP plasmid as your destabilized eGFP source, why don't you use that plasmid for cloning in your E2F1 promoter? That plasmid has only a minimal TATA promoter, which you can also remove by EcoR1 digest.
I personally prefer d2EGFP over Luciferase... But you might need to establish a stable cell line overexpressing your reporter.
Cheers,
Minna
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#6 Penguin

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Posted 23 June 2009 - 04:04 AM

If you have the original pTAL-d2EGFP plasmid as your destabilized eGFP source, why don't you use that plasmid for cloning in your E2F1 promoter? That plasmid has only a minimal TATA promoter, which you can also remove by EcoR1 digest.
I personally prefer d2EGFP over Luciferase... But you might need to establish a stable cell line overexpressing your reporter.
Cheers,
Minna


No, I have GFP fused to a mouse ornithine decarboxylase sequence which gives it a half-life of only about 30 minutes. It is in a vector called pMAZ and has a CMV promoter upstream. My E2F1 promoter is in a pBS plasmid but none of the restriction sites in the MCS are available for cloning in my dGFP - I'm going to have to do some PCR mutagenesis I think.....

#7 Rsm

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Posted 23 June 2009 - 05:50 PM

You got a PM...

Cheers,
Minna
I got soul, but I'm not a soldier




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