I am performing agarose gel electrophoresis of plasmid which is 2.5-3 kb in size. I am getting more than 3 bands, after restriction I got single band at 3.5kb in size. Electrophoresis conditions- 0.5X TBE, constant voltage 150V. why am I getting more than 3 bands after agarose gel electrophoresis of plasmid?
1$\begingroup$ Please convey the question in the title $\endgroup$– rg255Sep 29, 2016 at 15:27
$\begingroup$ It is probably due to the presence of supercoiled plasmid. You can try to heat up your plasmid in a thermocycler , 90 C for 20 min, this will denature the proteins responsible for the supercoiled structure and you will eventually see only one band representing the circular plasmid. The fact that you see only one band after digestion it's a good sign that you indeed have only one plasmid in your prep. $\endgroup$– alec_djinnSep 30, 2016 at 8:34
$\begingroup$ Thank you for reply. I am using sure spin column for extraction of plasmid. how does protein present in sample? $\endgroup$– Sonam PatilOct 1, 2016 at 4:04
$\begingroup$ @alec_djinn Is it known which bacterial proteins are responsible for maintaining the supercoils in purified plasmid DNA? $\endgroup$– RosieFOct 3, 2016 at 23:41
$\begingroup$ @RosieF specific DNA gyrases are responsible for inducing the coiling, then histone-like bacterial proteins keep the structure in place. Have a look here en.wikipedia.org/wiki/Bacterial_DNA_binding_protein and here ncbi.nlm.nih.gov/pubmed/26928284 $\endgroup$– alec_djinnOct 4, 2016 at 5:37
The closed plasmid comes in three different forms: nicked-relaxed, supercoiled and circular single-stranded. All three forms have a different tertiary structure and thus they run differently on the gel. This should look like this (image from here, there you also find a more detailed explanation):
When you digest the vector you linearize all the forms and they all have the same tertiary form.