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Cloning two genes in the same plasmid


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#1 saad

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Posted 06 February 2012 - 06:54 AM

Hi all,

I would like to try co-expression of two genes in one plasmid under different promoters.
I'm looking to pFast bac Dual plasmid then I've seen two promoter p10 and pPH with two MCS, and they are at opposite direction.
I don't know if the expression will be into two directions. I mean how to choose the restriction enzymes (which one, forward and reverse with the other promoter) they seem to me in opposition.
Could you please help.

Thanks

Edited by saad, 06 February 2012 - 07:13 AM.


#2 scolix

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Posted 06 February 2012 - 09:57 AM

the promoter drives the expression. if each gene's promoter will drive its expression. As long as the respective promoter and gene are in the right orientation, its fine.
Choose enzyme sites accordingly.

#3 saad

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Posted 07 February 2012 - 05:52 AM

Thanks scolix for that
I meant that if the arrows in plasmid's map are pointed to the opposite directions, does that show the orientation of expression.

#4 scolix

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Posted 07 February 2012 - 09:21 AM

the direction of arrow would indicate orientation of expression. but remember if someone made the vector map, they could also have labelled it the wrong way. So beware of it.

#5 saad

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Posted 07 February 2012 - 09:38 AM

Thanks a lot
that is very helpful
cheers




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