Promoter/gene cloning question - (Jul/23/2009 )
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?
anubis2005 on Jul 23 2009, 07:21 PM said:
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.
Could one use gateway cloning to get around this problem?
anubis2005 on Jul 23 2009, 09:03 PM said:
I suppose, but I've never done gateway cloning. Directional cloning isn't much of a problem, though. It's pretty simple.
Hi there,
If you don't mind can you make the question more simpler. A part of it is not making any sense
MS
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
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
anubis2005 on Jul 24 2009, 04:30 PM said:
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.