Hello,

Is it non modified pET28a plasmid ? How many restriction site have you for EcorI and Xhol ? One or twho ?

Maybe your plasmid is not fully digested and you observed your entire circular plasmid which migrates differently than your linear digest plasmid if you have only one restriction site. Also, I think that your lower band is your digested band.

For your uncuted plasmid, I think you have 4 bands because your plasmid is circular.

I hope it help you,

Colin

Are either of the potential digest fragments the size you expected? If not, I agree that its likely the plasmid wasn't digested fully and you're seeing different plasmid conformations (nicked, linear, coiled, supercoiled).

Alternatively, while some dna is still in the wells, could the second band be your plasmid in your digests? Expected size of the plasmid is ~7100bp; what marker are you using?

I think plasmid digestion is incomplete that is why you are getting two bands after digestion with single enzyme

Thank you all for your answer.

I have one resteriction site for EcoRl and XhoI. And ladder is 1kbp.

I also think digestion is incomplete. But with every enzyme I tried, I got the same result. I really do not know what the problem is.

@Noorul-Ain @Colin_Salauen @Hugh-White-2

Hello, May I know how you resolved your issue? I am also facing same problem but in my case the enzyme I am using is Dam and Dcm sensitive. I am suspecting I should grow them in a specific bacterial strain before isolating plasmid. Thank you Shiva Khoshkhoo