If you see only two bands on your gel, how can you tell the plasmid's conformation? For example, it could be supercoiled and linear, or it could be linear and nicked circular or concatamer...
This is my plasmid DNA bands. The size of my plasmid is 6200. I used a 1Kb ladder. In which all concatamer band is present. How to avoid concatamer? Any advice here?
If the next step is opening the plasmid then you do not need to know the conformation. Cut the plasmid with a restriction enzyme (s) appropriate to your cloning and then everything will be linear
Uncut plasmids do not run "true to size" on agarose gels.
You have nice bands on the gel, just continue with your experiment.
Good luck!
I used this plasmid for transfection. Can I use this for transfection? I need supercoil DNA for transfection. The band seen in the picture is it band for supercoil DNA or a concatamer?
Try looking at the existing literature.
Article Improvement of transfection efficiency by using supercoiled ...
To add to the excellent answers above, linearized plasmid runs with similar mobility to nicked circle (not supercoiled) whereas supercoiled plasmid runs faster. So you can take a little DNA and run side by side uncut with a sample that was cut by a restriction enzyme cutting only once. That will confirm to you that your DNA is mostly supercoiled.
Generally concatamers is only a problem if the host strain is RecA+, most molecular biology strains that people use these days are RecA- so you should not get concatamers. However if you transform a RecA- strain with a dimer for example, it will always give you a dimer form plasmid prep.
It does look to me (if I am reading the ladder correctly) that your first lane (farthest from the ladder) is probably the right size for 6kb range and the other plasmids seem about double that.
Hello Fatima,
To check if the plasmid you are dealing with is supercoiled, I would run two reactions. The first will be completely undigested (put this in lane 2 right next to the ladder in lane 1). The second will be digested by a single enzyme and put in lane 3 (check the plasmid map to ensure that the enzyme you choose does not have multiple restriction sites). The reaction in lane 3 should be the linearized plasmid DNA. If this matches up with the actual DNA size, a double digest is likely giving the correct result.
At first glance, your plasmid looks OK, 95% supercoiled. I do not recommend loosing time for further tests. Eventually, you can use DNase I instead of restriction enzyme. The nicked (circular) form containing SSBs will display the slowest migration. The linearized form (containing a double strand break) will migrate between the supercoiled and circular bands. The ratio in different bands depends of the DNase I amount and the incubation time.
Plasmid DNA should be cut using restriction enzymes so that it becomes linear since nicked DNA show slowest migration. You then use a 1kb ladder as a comparative tool when performing the the gel electrophoresis
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