Genecloning three types of molecules? - (Oct/08/2011 )

Hey!
Have gotten a question that its theoritcally
during genecloning with plasmid and bacteria with DNA fragment that
you can get three types of DNA molecules.

One type is hopefully a recombinant molecule containing
a DNA fragment that you want to ligase into the plasmid but
what are the other two types of molecules you can get
or am i misunderstanding the whole question

and how can you select these types of molecules?

ty

You are barking up the wrong tree - all three are the recombinant molecule, think about states of DNA.

bob1 on Sun Oct 9 06:35:13 2011 said:

I found this question and wonder too what the answers would be.

Why do you say all three are recombinant molecules?

Do you mean that because of the fact you treated the DNA it becomes recombinant anyway (even if there is no insert or even if its not cut), its just recombinant DNA because you took it out of the host ?

Or what do you mean?

The way I see it:

option 1: Plasmid DNA contains "wanted" DNA, thus being your recombinant
option 2: plasmid DNA was cut, but has no "wanted" DNA in it ==> a) either "empty" recombinant (but I wouldnt call it a "recombinant" molecule) or the "wanted" DNA is in it backwards (I would call this a recombinant).
option 3: plasmid DNA was never cut and is just the way it was before (I wouldnt call this a recombinant, and its a bit the same as option 2a)

Or am I missing something here?
(this questions seems more about semantics/use of words)

Think about plasmids and the forms you see on a gel...

bob1 on Mon Oct 10 00:49:16 2011 said:

I still dont get it.

Do you mean that you only start working with plasmids that have been cut? But how do you know they have been cut? You use only the piece of the gell with the correct lenght, but you cant tell its been cut or not? Or can you?

Does a gel only contain straight pieces? Then what happens with the circular ones that have not been cut?

I think bob1 could be talking about plasmid DNA supercoiling and so on... and how DNA runs on a gel when it has not been digested (you do not see only one band).

If not, I would think of the three options that have been given by Lyok!

OA17 on Mon Oct 10 14:53:57 2011 said:

Do you mean that the uncut DNA will be shown in more then one band? (I cant imagine this, I mean: its just all the same, uncut, DNA?)

Or you mean that you will see 1 band for the cut DNA and 1 for the uncut DNA?

(not taking in account possible other bands because of wrongly cut DNA)

Yes, what I mean is that when you run an uncut plasmid on an agarose gel, you will not see only one band, because DNA forms different supercoiled structures (DNA topology and that stuff). When you digest plasmid DNA you eliminate the forces that make it supercoil and that is why it runs giving rise to a single band (if you use a restriction enzyme that only cuts once, or more bands, depending on the enzyme you use and the restriction sites). But of course, uncut plasmid DNA gives more than one single band.

I am not an expert, so I can be making a mistake, but I think this is what bob1 was referring to. Anyway, this is easy to be tested by yourself!

(By the way, sorry for my English!)

Yup, supercoiling etc. is what I was referring to. I was being deliberately obtuse as I had a pretty strong impression that the original question was homework.

OA17 on Mon Oct 10 20:18:41 2011 said:

bob1 on Mon Oct 10 22:09:26 2011 said:

Ok I see what you guys mean.


There is however one thing I do not understand.
The definition I always learned is the following: a recombinant molecule/DNA is DNA that contains DNA that is normally not present there. Or: a recombinant molecule is a man made combination of 2 (or more) molecules/DNA and is not present in nature.

Now: to me a uncut plasmid or even a cut plasmid is not really "man made".. And for sure it does not contain strange DNA.. So I find it weird to call that a recombinant molecule because its nothing weird or really man made.

So isnt this question more about semantics?