Hi there,

Analysis of the problem would be easier with a gel picture.

Do you know the restriction map of the plasmid? In other words do you know what is the expected digest profile of the plasmid?

For your info, an uncut plasmid already exhibits a multiple band pattern on agarose gel and if using too much of EcoRI enzyme it is losing its specificity (star activity) and gets able to cut elsewhere in DNA and XhoI shows lower activity on supercoiled DNA.

One or both enzymes cut your plasmid many sites thats why you see multiple bands.Copy your plasmid sequence and paste it in NEB-cutter or Web-cutter or addgene to see the sites. Plasmid is properly digested or not, that you can confirm from a apt. gel picture.

Hi there,

Analysis of the problem would be easier with a gel picture.

Do you know the restriction map of the plasmid? In other words do you know what is the expected digest profile of the plasmid?

For your info, an uncut plasmid already exhibits a multiple band pattern on agarose gel and if using too much of EcoRI enzyme it is losing its specificity (star activity) and gets able to cut elsewhere in DNA and XhoI shows lower activity on supercoiled DNA.

Thank you all friends and fellow to solve my problem. I did successful digestion of plasmid. As i came to know that there was probelm with digeston time and enzyme concentration. I used enzymes 0.5ul for  1, 2 ,3 and 4 hours. I got succesful results on 1 and 2 hours digestion.