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Promoter/gene cloning question


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7 replies to this topic

#1 anubis2005

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Posted 23 July 2009 - 04:19 PM

Hi,

I have a question about the manner in which a gene becomes transcribed under the promoter of a plasmid. Wouldn't the template strand have to be on the same strand as where RNA polymerase binds to on the promoter? How do you ensure this happens?

#2 fishdoc

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Posted 23 July 2009 - 05:48 PM

Hi,

I have a question about the manner in which a gene becomes transcribed under the promoter of a plasmid. Wouldn't the template strand have to be on the same strand as where RNA polymerase binds to on the promoter? How do you ensure this happens?




Yes. Directional cloning. Find a restriction site near the promoter and use that at the beginning of your insert. Alternatively, you can clone into one site and theoretically 50% of the inserts will be in properly.

For directional cloning, if you had a promoter followed by ecoRI, xbaI, notI, sacI, and sphI, you would amplify your gene with an ecoRI site engineered into the 5' primer and sacI (or xbaI, notI, sacI, or sphI) engineered into the 3' primer. That way the construct can only go in one way.

#3 anubis2005

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Posted 23 July 2009 - 06:03 PM

Could one use gateway cloning to get around this problem?

#4 fishdoc

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Posted 23 July 2009 - 06:41 PM

Could one use gateway cloning to get around this problem?



I suppose, but I've never done gateway cloning. Directional cloning isn't much of a problem, though. It's pretty simple.

#5 miraclestrain

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Posted 23 July 2009 - 07:28 PM

Hi there,

If you don't mind can you make the question more simpler. A part of it is not making any sense

MS

#6 Rsm

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Posted 23 July 2009 - 08:22 PM

Hi,
Your gene has to be in the same direction as the promoter. Most expression plasmids have a multiple cloning site downstream of the promoter, where you can insert your gene using two different restriction enzymes (directional cloning).
Cheers,
Minna
I got soul, but I'm not a soldier

#7 anubis2005

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Posted 24 July 2009 - 01:30 PM

A gateway cloning expression vector question


How do you know your gene will be transcribed and translated using the correct strand of DNA:

For instance: You don't want this

TATATAA..ATGCTCCCG
...............TACGAGGGC


you want this

TATATAA..TACGAGGGC
...............ATGCTCCCG

Right? You want the template strand to be on the same strand of the plasmid where the RNA polymerase binds to.


How do you know which strand the RNA polymerase will bind to, and how do you ensure your template is on that strand

#8 fishdoc

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Posted 24 July 2009 - 01:56 PM

A gateway cloning expression vector question


How do you know your gene will be transcribed and translated using the correct strand of DNA:

For instance: You don't want this

TATATAA..ATGCTCCCG
...............TACGAGGGC


you want this

TATATAA..TACGAGGGC
...............ATGCTCCCG

Right? You want the template strand to be on the same strand of the plasmid where the RNA polymerase binds to.


How do you know which strand the RNA polymerase will bind to, and how do you ensure your template is on that strand




If you're saying TATATAA.. is written 5' to 3', then the first one is what you want, not the second one. In your second example, if the top strand is 5' to 3', then you bottom strand 5' to 3' would be GCCCTCGTA, not ATGCTCCCG.


The RNA polymerase binds to the antisense strand to make an RNA copy of the sequence. The mRNA is the same sequence as the DNA strand (the strand on top, the sense strand), except for U substituted for T, of course. So in your example, the mRNA sequence would be AUGCUCCCG.

Edited by fishdoc, 24 July 2009 - 01:58 PM.





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