We recently isolated plasmid DNA with a new kit, and when we analyzed it on the agarose gel - in addition to the major plasmid band, we saw some DNA aggregates stuck in the well and smearing. When we repeated isolation and tried all standard tricks like intense pipetting, vortexing and heating- the band was still there. Any idea what this might be? how to resolve it? thank you!
Dear Alexander,
I think that the other bands present on your agarose gel well may be RNA contamination thus vortex and heating will not help. Also, in addition to the expected band that are present on the gel, the other bands plasmid DNA may be due to the other forms of plasmid DNA (B DNA, A DNA or Z DNA forms), But they are distinct on your gel because of the smearing caused by the RNA contamination with your extracted DNA. There is always a high chance to have all 3 forms of the of plasmid DNA at the same time during DNA extraction though the B form is the majority form DNA. However, it is also possible to have only one band on your agarose gel. So you can try to resolve your extraction problem by trying the extraction Kit "Wizard Plus SV Minipreps DNA Purification System" from Promega. I used this Kit for my plasmid DNA extraction and the result looks excellent and I still have the 3 bands described above. Hope I have tried to help you with your problem.
You may have a concatemeric situation on your hands.
http://www.sciencedirect.com/science/article/pii/S2001037014000269
Thank you Marius and Jack. this is not an RNA aggregate- as RNase treatment did not help (and RNA fragments usually migrate towards the bottom of the gel). does not seem to be a protein complex- as proteinase K treatment also did not eliminate this band stuck in the gel well. Not misc DNA forms for sure. should not be any concatemers- as these are simple, well known plasmids that we purified earlier without any problems. I am puzzled.........
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