When purifying my pET28a plasmid using a commercial kit, the Nanodrop test gave me results of 500 ng/µL. However, when I verified that it was plasmid in the 1% agarose gel, I added 2,000 ng of plasmid DNA to the well. The result is as follows (image).
The first well corresponds to a plasmid digestion with NdeI and NotI.
The second well corresponds to my pET28a control (2000 ng).
At the end of the gel, degradation of genetic material is observed.
One option for the problem.
- The kit - Solution A containing RNAse - this enzyme is degraded.
Gel agarosa 1%
Buffer TAE 1X
VOLTAJE: 80 V
Tiempo: 35 minutos
What should the intensity of the 2000 ng plasmid band look like?
The nano drop will tell you the concentration of nucleotides, but not whether they are in a single DNA molecule or degraded.
What you should see is a single, very bright band at a large size. Based on your image,I don't think your plasmid extraction was successful.
Double check that you grew your cells in the right selective media. That's the most obvious way to have an error.
Good luck!
I agree with Katie A S Burnette that your purification was not successful. If you look at the plasmid lane, in addition to the faint plasmid band you also see a large smear through the middle and then a very bright spot at the bottom that could either be degraded DNA or RNA. Nanodrop will measure all nucleic acids which is why you have a high reading.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology