Hi all,
I used a ZymoPURE II Plasmid Maxiprep kit to isolate plasmid DNA and ended up with very low yield and poor quality. Specifically, less than 10ng/uL in 500uL from 150mL of culture, and 260/280 ratios of 0.75-1.5. I have used this kit many times before and always gotten good yields/quality, including when expressing and purifying the specific plasmids I was attempting.
I believe the problem had something to do with the binding buffer addition, as prior to that everything was as expected/prescribed by the ZymoPURE handbook-the resuspension, lysis and neutralization steps all had the standard color and consistency changes, resulting in a clear yellow clarified lysate after being passed through the Syringe Filter-X. However, immediately upon addition of the binding buffer the lysate turned slightly pink and cloudy, similar to how it looks when you initially resuspend the pellet in P1 buffer, but less opaque and the pink more dilute/lighter and with a grayish tint. The flow-through from the Spin V-PX column was still pink, but not cloudy, and the washes and final elution were clear.
Does any one have any insight on what might have happened?
Thanks for your help!
Sarah
I had the same experience yesterday; I got a very low copy DNA yield after the plasmid purification. I examined the DNA concentration in the binding buffer, which was higher than in the elution.
I need recommendations.
I also faced same problem. I am also looking for solution. I appreciate your suggestions. Thank you.
- Badges
- Science topic
- Similar topics
- Biological Science
- Microbiology
- Virology
- Virus