I was wondering if I could use ethanol for DNA extraction for running a gel. Any precautions? Tips?
This is the protocol I was thinking of:
Pipet out the supernatant and combine pellets to centrifuge in new tube again at 16000 X g for 3 minutes to ensure absolute purity of pellet.
If any supernate is seen pipet out. (keep on ice when not on centrifuge)
In a centrifuge tube place the pellet on ice to keep cool. Then pipet ethanol onto the solution and continue until one third of volume is ethanol (1:3) .
The supernate is to pipetted into a new tube and spun at 16000X g for 3 minutes. (keep on ice)
The supernate is to be pipetted off and the pellet is to be removed into a new tube. (keep on ice)
To remove ethanol, place in Protein Collection Tube and spin at 15000X g for 15 minutes. Discharge supernatant and retain pellet. Wash pellet in ethanol (1:9 ratio) vortex and repellet sample.
Thanks!
THIS IS SO SIMPLE: JUST INVERT THE TUBE ON A CLEAN TISSUE TO AIR DRY FOR MINUTES. ETHANOL INTERFERES WITH PCR THEREFORE, IT MUST BE COMPLETELY REMOVED AND STORED.
People precipitate DNA with ethanol and run it on a gel all the time. What you describe sounds a little atypical, but I'm not sure what sort of sample you are starting with. Things to keep in mind 1) After initially precipitating DNA in 100% EtOH, washes should be done in 70% EtOH 2) DNA pellets are usually tiny and I would avoid moving them between tubes if possible. Its best to combine all your DNA when it's still in aqueous solution, add the EtOH (and salt if needed), and then spin. Keep the pellet in this tube for one or two additional washes 3) At the end, remove all the liquid you can and then air dry the pellet. I usually do this for 10 to 15 minutes 4) Last, you need to get the DNA back in solution by adding TE or water to the pellet. For small amounts of DNA, you can immediately pipet to get the DNA resuspended. Then you can combine some of this with loading dye and run it on a gel. If you don't get rid of most of the EtOH, the sample won't sink into the wells.
Thanks I was concerned that I would mess this project up (its a science fair for my Community College). I truly appreciate your input and you relieved my concerns about using ethanol. I would imagine that using 95% ethanol would be alright? In the lab we don't have anything higher than that. I know that we will have some 70% once I make it. :)
Many thanks!
Micah
I would to suggest 2-Propanol (Isopropanol) for DNA precipitation, coz if your DNA sample volume is too it leads to very low concentration. The advantage in using 2-Propanol is to get even too low concentration and intact DNA too. Its just a suggestion.
- Badges
- Science method