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Genecloning three types of molecules?


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11 replies to this topic

#1 JellowK

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Posted 08 October 2011 - 04:46 AM

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

#2 bob1

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Posted 08 October 2011 - 10:35 PM

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

#3 lyok

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Posted 09 October 2011 - 09:42 AM

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


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 B) 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)

Edited by lyok, 09 October 2011 - 09:44 AM.


#4 bob1

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Posted 09 October 2011 - 04:49 PM

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

#5 lyok

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Posted 10 October 2011 - 01:24 AM

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

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?

#6 OA17

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Posted 10 October 2011 - 06:53 AM

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!

Edited by OA17, 10 October 2011 - 07:01 AM.


#7 lyok

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Posted 10 October 2011 - 10:30 AM

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!


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)

#8 OA17

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Posted 10 October 2011 - 12:18 PM

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! Posted Image

(By the way, sorry for my English!)

#9 bob1

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Posted 10 October 2011 - 02:09 PM

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.

#10 lyok

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Posted 10 October 2011 - 10:44 PM

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! http://www.protocol-online.org/forums//public/style_emoticons/default/wink.png

(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.


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?

Edited by lyok, 10 October 2011 - 10:51 PM.


#11 OA17

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Posted 11 October 2011 - 05:29 AM

What I understand from the original question is that you have already cloned the insert, and so you already have the recombinant plasmid. Any uncut plasmid (recombinant or not) would give several bands on a gel.

However, if you are testing the result of the ligation by analysing your colonies, you can have these results:

- Plasmid containing the desired insert. (Congratulations!). Recombinant plasmid, for sure.
- Religated plasmid (even if you used different restriction enzymes, religated products do sometimes appear, although if you treat with a phosphatase after digestion, this should be avoided).
- Original plasmid: because of incomplete digestion and religation (or no digestion at all) and you purified it together with the digested plasmid.

Since we generally clone into plasmids that have already been engineered for this purpose (think about multicloning sites, promoters, antibiotic resistance genes, etc) I would say all of them are recombinant.

To bob1: Yes, it looks very much like a homework question...!! perhaps I should not have answered.... but it will be his/her task to apply his criterium and believe what we have said, or not. We can always be wrong! ;-)

#12 lyok

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Posted 11 October 2011 - 07:38 AM

What I understand from the original question is that you have already cloned the insert, and so you already have the recombinant plasmid. Any uncut plasmid (recombinant or not) would give several bands on a gel.

However, if you are testing the result of the ligation by analysing your colonies, you can have these results:

- Plasmid containing the desired insert. (Congratulations!). Recombinant plasmid, for sure.
- Religated plasmid (even if you used different restriction enzymes, religated products do sometimes appear, although if you treat with a phosphatase after digestion, this should be avoided).
- Original plasmid: because of incomplete digestion and religation (or no digestion at all) and you purified it together with the digested plasmid.

Since we generally clone into plasmids that have already been engineered for this purpose (think about multicloning sites, promoters, antibiotic resistance genes, etc) I would say all of them are recombinant.

To bob1: Yes, it looks very much like a homework question...!! perhaps I should not have answered.... but it will be his/her task to apply his criterium and believe what we have said, or not. We can always be wrong! ;-)


Ah yes, I didnt concider the fact that most of those plasmids are allready altered for our use.




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