After double digestion of my plasmid DNA with EcoRI and HindIII I have obtained two bands as expected. But when I digested the plamid DNA only with EcoRI to linearize it, I obtained three bands including one expected. The two additional bands are probably not uncut plasmid forms (I run uncut plasmid DNA alongside) and are smaler then the expected band. Conditions, components and its concentration in the second reaction was the same as in the first. What is the explanation of observed effect? Which of results is reliable (from the double digestion or from the single digestion)?
What are your band sizes? Add them up and see if they are the same total number of base pairs. You might also be seeing uncut plasmid - which can run as multiple bands (supercoiled, relaxed, nicked, etc.).
I agree with Katie A Burnette, the other forms of plasmid (supercoiled, relaxed, nicked, etc.) may run more quickly than the digested form.
i think your EcoRI may has cut only partially the plasmid, but when you made double digestion your plasmid was completely digested.
I agree with Katie A Burnette and i think your EcoRI may not work at all. please indicate if your plasmid is Native or it has a gene in it.
Its probably your EcoR1 is not working probably, leaving some plasmid uncut, or making nicked. Nicked plasmid run very slower than digested, where as uncut will run faster.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Enzymology
- Enzymes