Why do you need your insert in the vector in both orientations? - Cloning (Aug/02/2007 )
I am a cloning "newbie" and I have a question that maybe some of you can explain to me.
When cloning, why is it sometimes important to have both orientations of an insert in the vector? From what I understand, sometimes you only need one orientation, which is the one where the 5' is by the promoter, which will give gene expression. however, I also heard that some will want both orientations to study (e.g., they may want to study antisense(not sure what that is other than the 3' region?)).
Could someone please explain this in greater detail for me.
Also, how can you tell from a restriction enzyme digest alone what orientation you have?
Thank you!
At the moment I can't think of a reason.
But while I think of one, orrientation of an insert can be determined if the restriction enzyme cuts the insert asymetrically, so that one half (perhaps the 5') is longer then the other half.
Now a second cut is made in plasmid, this time on the vector backbone, at the know position. This cut may be by the same enzyme or another.
With 2 cuts sites, the plasmid is cut into 2 fragments. Where one cut site is at a known position. If we know the structure of the desired plasmid, we will know the distance between the two cut sites. (aka the fragment size). Since the insert was cut asymertically, the restriction site in the insert can be either closer or futher away from the cut site in the vector. Dfferent distances between cut sites, in turn means that the fragments cut out will be of different sizes. So if you know which fragment size to look for, you can determine the orientation of the insert in the vector.
Some people might use the wrong orientation insert as control.
But while I think of one, orrientation of an insert can be determined if the restriction enzyme cuts the insert asymetrically, so that one half (perhaps the 5') is longer then the other half.
Now a second cut is made in plasmid, this time on the vector backbone, at the know position. This cut may be by the same enzyme or another.
With 2 cuts sites, the plasmid is cut into 2 fragments. Where one cut site is at a known position. If we know the structure of the desired plasmid, we will know the distance between the two cut sites. (aka the fragment size). Since the insert was cut asymertically, the restriction site in the insert can be either closer or futher away from the cut site in the vector. Dfferent distances between cut sites, in turn means that the fragments cut out will be of different sizes. So if you know which fragment size to look for, you can determine the orientation of the insert in the vector.
Thank you for replying to my question in such a timely manner!
What you say makes sense to me now. If I do a restriction enzyme digest using one enzyme that cuts once at a specific place in the vector, I should see one band that is equal to the size of the insert plus vector; however, I may see two bands if there is that same site within the insert. Therefore, if there are two bands, it can be determined which orientation the insert is in the plasmid by examing the sequence of the insert. For example, if the insert is 3kb adn the vector is 2 Kb,and the restriction site is at 1kb of the insert and the other restriction site in the vector is just 1 kb downstream from that, I should expect one band to be 1kb and the other band to be 4kb. If the insert is in the other orientation, however, I should expect to see one band at 2kb and the other band at 3Kb when I run my enzyme digest. Is that correct?
So, I feel foolish asking this (it has been awhile since I've been in school) but someone give me the "run-down" of the importance of orientation and how it plays into gene expression. I just know that the 5' must be at the end of the promoter...
One more question (sorry), is the pENTR3c vector an expression vector? Years ago, I would clone genes into a "cloning vector" and then cut and re-ligate into an expression vector. So, is this pENTR3c a new technology where you can just clone right into it and have expression?
Anitsense constructs are useful controls for things like transfections. If you just use empty vector to transfect, the control plasmid will of course be of a different size. So you're then left with the question, "Should I use equivalent nanomoles of control DNA or equivalent nanograms". Whatever you chose to do, it is an incontivertible law that at least one of your reviewers with argue that you should have done the opposite.
You might expect an anitsense construct to in theory produce a completely different protein because the code is being read backwords. In fact though, if you flip just about any DNA sequence around your antisense gene will have numerable stop codons introduced into the sequence- prohibiting the synthesis of a protein even if translation was initiated