To remove STOP codon I've cut my plasmid, blunt-ended (actually I'm going to do that) and ligated to itself.
The question is, in case that two molecules of plasmid would join together (in the same orientation) and form sort of dimer, how could I distinguish that from a single plasmid molecule with intramolecular ligation? In case I'd wanted to do restriction analysis, it will cut twice and I would get fragments of the same and correct size anyway. Similarly for PCR.
The only idea I have is to compare actually uncut plasmid on gel with the original, because it should be slower because of it's size (of course not proportionally) but I should be able to see some difference from the original plasmid?
My best guess would be to run the original uncut plasmid side by side with what you think is your output.. Both uncut. The uncut and supercoiled sizes should be the same if they are the same size -- if it is now effectively 'doubled' it should run differently. I'm not absolutely sure this would work, but that's the best thought I could produce in 1 minute of brainstorming.
Dear Thomás, It can be very difficult to differentiate a monomeric DNA sequence from a dimeric form, other than by apparent uncut sizes or band intensities after digestion. However, practically this is not usually required as the host bacterium is most likely to resolve the dimer at a very early stage and produce monomers. I would suggest that even if your ligation produced a significant proportion of dimers compared to monomers, by the time you have grown up cultures from transformant colonies, all you will detect are monomer forms of the plasmid (even if you are using a recA strain). Regards, Andrew
Hi, the above answers are absolutely fine and should solve your problem. But, still if you really want to put effort to know if there are any concatemers...you can design overlapping primers that will give pcr product only when two DNA fragments are joined. forward primer should start at the end of first plasmid and reverse primer should end in start of second plasmid molecule. this will surely work. all the best!!
I agree with the above answers. The bacterial cell will resolve the dimers, and also create new ones IF the strain is Rec+ (which not all are). If you are using rec- cells then there will be no recombination either larger or smaller. You can compare uncut plasmid sizes from mini preps from rec- cells.
Having said that, Why do you care? Wild type cells will create dimers, trimers, tetramers etc, those are fairly natural. So it may not matter.
the simplest way to do it is use a different restriction enzyme. If you have the somplete sequence of your plasmid you could simulate the monomer and the dimmer and check for a restriction enzyme that gives you some differential band when comparing both plasmids.
I will have to cut at leas 500 pb away from you ligation site to make it easy to determine the different band.
It could be a good idea to use an enzyme that cuts multiple times in the plasmid so you can have a nice pattern instead of one or two bands that can be confused with uncut plasmid.
That works if you have a head to head dimer (the molecules are inverted with respect to each other). But if you have a head to tail dimer (directly repeat) then it won't.
All great answers.
Additionally, you can limit the formation of concatenated plasmids by lowering the concentration of DNA during ligation. It is then much lesslikely that a given molecule "meets" another one (as opposed to self-ligating, which does not depend on concentration).
If you can identify conditions for a partial RE digest, such digest would give you three bands in case of a dimer (uncut, single cut, two cuts).
Restriction digest could possibly be used to identify the dimer even if you have an enzyme that will produce a single cut in the monomer plasmid. If you make a diagram, you will find that a single cut on the monomer will produce one linear 5 kb fragment. However, the same enzyme will likely cut the dimer in two places with products of different size. This would work for a dimer formed head to tail or head to head.
A dimer composed of two plasmids each with its own ori (origin of replication initiation) will not replicate in lab strains of bacteria, so do not worry about it.
HI it may be late now but incase you haven't tried just cut with one single cuter enzyme and if its dimer you will see double its size.
Hi Cody you are absolutely right (I forgot that they are exact copies) and your suggeston about running uncut plasmid will difinetly show the differences between the two.
Also one can do a simple inverse PCR and get the differences in product between the two.
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