I tried many times for the extraction of plasmid(PET28a vector) . The concentration is very high. But the problem is after running Gel electrophoresis i get 3 bands as you see the attached picture.How i can get PET 28a vector Single band on the agarose gel? Your suggestions will be highly appreciate.
hi
In plasmid extraction by alkaline digestion, you should mix gently by inverting the tube(4-6 times ) . dont vortex. Staying over 4 minutes in this buffer(200 mM NaOH + 1% SDS (w/v) )will cause the plasmid to be broken down.
good luck
It's quite normal for a circular plasmid prep to look like that because there are a few DNA forms generated during DNA extraction (circular, 'nicked' and linear for example). For most applications, it doesn't cause problems. If you circularize your prep using a restriction enzyme, you'll normally see a single band running true to size.
Supercoiled, Semirelaxed and relaxed will give three different sizes. So I agree to Pavels explanation.
BTW for Transfections: The more supercoiled plasmid you have th ebetter it gets transfected into the target cells
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running