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 $\endgroup$ – rg255 Sep 29 '16 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_djinn Sep 30 '16 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 Patil Oct 1 '16 at 4:04
  • $\begingroup$ @alec_djinn Is it known which bacterial proteins are responsible for maintaining the supercoils in purified plasmid DNA? $\endgroup$ – RosieF Oct 3 '16 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_djinn Oct 4 '16 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): enter image description here

When you digest the vector you linearize all the forms and they all have the same tertiary form.

| improve this answer | |

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.