Troubleshooting for DNA purification - (Feb/15/2012 )
I have just started working in a microbiology lab at my university, and the grad student I am working under has given me a variety of simple laboratory tasks to complete such as DNA purification, PCR, agarose gel electrophoresis, etc. I am currently having problem consistently producing quality DNA preps, with the low end coming out at 6-7 ng/microliter and the good preps coming out around 300 ng/microliter. The problem is I don't particularly understand what I am doing correctly, and what I am doing incorrectly.
The lab uses the Promega Wizard kit for DNA purification, and the Gram-negative bacteria protocol. So, the steps in descending order are:
1. Centrifuge overnight culture in PBS solution for 2 minutes at 13.2K RPM then decant
2. Homogenize pellet via gentle pipetting in nuclei lysis solution, then incubate at 80 degrees centigrade for 5 minutes, and allow to cool
3. Add RNase and incubate at 37 degrees centigrade for 45 minutes, then allow to cool
4. Add protein precipitation solution, vortex and incubate on ice for 5 minutes, then centrifuge for three minutes
5. Add supernatant to clean tube containing isopropanol, and mix
6. Centrifuge for two minutes, decant the supernatant, and add an equal amount of 70% EtOH.
7. Centrifuge for two minutes, then carefully aspirate the ethanol.
I believe I am doing the final two steps incorrectly, as the DNA pellet is invisible and I don't really know if I am taking some of it out when I pipette the ethanol solution away in the final step. I also may not be sufficiently resuspending the pellet after decanting the ethanol, or washing the walls of the tube with the ethanol.
In a DNA prep today, I made sure to carefully take off the ethanol away from the presumed location of the pellet, and I left a small amount at the bottom which covered the pellet slightly. This resulted in improved values of 119 ng/microliter this time. Ultimately though, I don't really know how to do this step correctly and would like to have some input. I have been looking for a video tutorial but so far none has been forthcoming.
You may (should) be able to improve the yield by precipitating in the IPA for longer and/or at a lower temperature (-20 C commonly), and spinning down the DNA for a longer time, which will also help attach the pellet to the tube wall.
The amount of DNA is also dependent on the starting amount of bacteria you have, it may be that you are starting with different amounts of bacteria, which is why you are getting wide variation. Having said that, you would need a tiny amount of bacteria to get 6-7 ng/ul.