Hello all,
I have a mini-prepped DNA that I am trying to digest with EagI. After doing the digestion it looks like the reaction with the restriction enzyme maybe linearizing my plasmid as it migrates at its expected weight when next to the DNA ladder. However the undigested sample runs very high, far above the 10kb band of the ladder. I am guessing this is the uncut is showing a nicked plasmid, which is why it's running so high? What do you think?
Thank you
MB
Hi, it is normal to see the uncut plasmid band above the cut plasmid band.
Assuming that the host strain you made the plasmid prep from is a recA- strain (most strains like DH5a etc are) then you probably are propagating a dimer or trimer plasmid that because there is no recombination, does not resolve to a monomer. I've seen this a few times. It should not be an issue and upon digestion everything will run at the correct size.
Normally a monomer supercoiled plasmid will run faster than linear and the nicked circle monomer species will run with very similar mobility as linear.
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