I did transformation for some plasmid into E.coli and spread it on agar with antibiotics, then i chose a colony and did DNA extraction (with miniprep) and then i ran the gel. for some reason i am not sure what is the sequence of the plasmid but i know it's size, so I am not sure which exact restriction enzymes should i use. when i ran the gels I got some bands at the exact size of the plasmid that i should have (which is 7.3 Kb). is that a good proof that i have the right colony with the the desired plasmid in it? can carry on with my experiments on that?
thank you all
I am not an expert but in my opinion you should run electrophoresis using sample without using restriction enzymes and other samples where you use it.
It would be perfect to know the sequence of the plasmid you're testing. Do you have anyone that you could ask about the sequence or to give you a little hint?
There are situations (when using wrong bacteria strains) when you obtain the same BPs numbers after transformation but the sequence is not the same.
No, you cannot assume anything based on what you have.
Never use a plasmid if you don't know EXACTLY what it is! And if you didn't digest the plasmid, then you can't rely on it running "true to size" on an agarose gel. Plasmids can supercoil and run larger than expected, or smaller than expected, or BOTH at the same time.
There are dozens of common plasmids commercially available, many of which are "about 7.3 kb" in size. Unless you personally ordered this plasmid from a company, you cannot know what you have.
Stop what you are doing, ask for help. Sequence your plasmid (even if someone promises you don't need to), and then continue your experiment.
Two weeks of investigating now will save year years of trouble.
Katie A S Burnette thank you
but i applied one uncut and one with one restriction enzyme (because it was the only enzyme i was sure that it was in the plasmid) to avoid supercoil and it looks like that the RE solve the supercoiling. what could be causing this band then? ( i am sure of the plasmid that i am adding, but i am not sure of it's exact sequence because it was constructed in my lab ten years ago)
could a contamination cause the same band ?
No, contamination doesn't make a 7.3kb band magically appear.
Stop what you are doing, sequence the plasmid. Trust me on this one.
I agreed with Katie AS Burnette, "contamination doesn't make a 7.3kb band" magically appear, it might somewhat show a thymine dimmer below the lowest band on the gel. sequencing the plasmid is the best way to determine whether you chose the suitable colony after transformation.
Best of luck brother.
The presence of bands at the exact size of the expected plasmid (7.3 kb in your case) in the DNA gel electrophoresis is a good indication that you have the right colony with the desired plasmid in it. However, it is important to consider additional factors to confirm the presence of the desired plasmid and ensure the success of your experiments. Here are a few points to consider:
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