We have recently encountered problems with our plasmid DNA minipreps. We ran
out of the particular DNA batch used for transfections and started making
more DNA by transformation of our home-made competent DH5alpha E.coli cells,
plating and starting overnight miniprep cultures from single colonies. The
DNA used for transformations has been sequenced, is clean and has worked
well in transfections. No extra bands are visible in this prep.
On agarose gel with undigested plasmid prep, we see our plasmid of ~ 6.7 kb
(pcDNA3 plus our gene in EcoRI/XhoI cloning sites) plus faint bands of ~ 8
kb and 10 kb. The extra bands (plasmids?) are digested by restriction
enzymes with the correct plasmid to produce some more faint extra bands. All
our DNAs have previously always been free from these extra bands.
We have checked the following things:
-picked several well-isolated colonies; the amount of 'extra' DNA varies
from colony to colony but is visible in each of them
-transfected competent cells using another DNA preparation but the 'extra'
bands are still visible
-bought a new miniprep kit; nothing helps
Do you have any suggestions how to overcome this problem and obtain clean
DNA for transfections? Your help will be very much appreciated,