4 replies to this topic

[clwop]

In mapping a plasmid, i digested it with BamHI and Hind III, resulting in 4 fragments, 1.9, 1.1, 1.5, and 4.6 Kb. On the gel the 4.6 Kb band  was less bright than the rest ones. it was really weird because  the brightness was supposed to be directly proportional to the amount of DNA present. And this kind of thing never happened before. What could be the reason? did i overdigested it? was it the gel's problem. I added ethidium bromide into the gel before pouring it, but i always did this way. can anybody help me?

[clwop]

[bob1]

You may have underdigested it - so some of it is still in plasmid form rather than linear fragments.

[clwop]

bob1, on 24 August 2011 - 05:13 PM, said:

You may have underdigested it - so some of it is still in plasmid form rather than linear fragments.

The three bands between 1 Kb and 2 Kb can't be all undigested plasmid. They are all brighter than the 4.6 Kb band. I just did this experiment again from plasmid isolation. This time the result is quite nomal.

Edited by clwop, 24 August 2011 - 11:28 PM.

[bob1]

Ok, perhaps one or more of the sites is blocked by something such as methylation, or happens to be partially mutated in your stock.