I am going to conducted a study that isolates the DNA from urine. Which method should I used to perform the experiment? I need to perform a DNA isolation method in the lab and not the prepared kit available on the market.
To perform the extraction of DNA from urine first centrifuge the urine, then incubate in buffer plus proteinase K and subsequently extract the DNA method with phenol / chloroform
I think phenol chloroform method will be the best way to extract DNA from urine.
Take 15ml-20 ml urine
centrifuge at 3000 rpm for 25 minutes
wash pellet with 50 μl of
digestion buffer (50 mM Tris–HCl, pH 8.5; 1 mM EDTA; 1 % Triton X-100 and 0.5 % Tween 20) + 200 μg/ml of Proteinase K
then use phenol chloroform method of DNA isolation
A protocol that I used with success is the following: Approximately 15 ml of urine are centrifuged at 3,000 rpm for 20 min; the pellet resuspended and washed three times with 1 ml of phosphate buffer saline (PBS), and then digested for 3–5 h at 56 °C in 150 μl of digestion buffer (50 mM Tris–HCl, pH 8.5; 1 mM EDTA; 1 % Triton X-100 and 0.5 % Tween 20) containing 200 μg/ml of Proteinase K. DNA is purified by phenol and phenol-chloroform-isoamyl alcohol (25:24:1) extraction and ethanol precipitation in 0.3 M sodium acetate (pH 4.6). (J Med Virol 2010)
Dear Abdul,
all the protocols below (and the ones to come) are good protocols and should work;
nevertheless I was wondering if for your application you couldn't try something "easier" and more light. I am thinking of adding magnetic beads to capture nucleic acids in the medium and to isolate by magnetic field.
This application reminds me the one we propose for capturing viruses in medium wihtout any specificity: there is no use of antibody or other rec prot on which the virus or the nucleic acid bind.
I never tested our magnetic nanoparticles in your application but I think it could be worth trying. May be we could figure something out.
just let me know if you want to try or need more info (you can reach me at [email protected])
good luck for your experiments,
Cedric
This is interesting, i have done it a couple of times. You can employ the Phenol-chloroform method. This is done by adding equilibrated phenol and shake vigorously. Then spin in a centrifuge at 16,000rpm for 3mins to separate the phases. Then aspirate the aqeous phase in a new eppendorf tube and add equal volume of chloroform. Again, shake vigorously and centrifuge to remove traces of phenol. Carefully aspirate the aqeous phase into a new eppendorf tube. Now is time to precipitate the DNA by adding 50ul 3M sodium acetate and 1ml of 100% alcohol into tube. At this point you shake vigorously and freeze at-80C for 40mins or -20C for 1hr. Centrifuge at full speed for 30mins at 4C and decant supernatant. Add 300ul of 70% alcohol to the DNA pellet and shake vigorously again. Decant the supernatant and air dry pellet at 37C for about 10mins. Re-suspend your DNA by adding 50ul of TE buffer and shake vigorously. Store at -20C until ready to use. My friend, enjoy your research.
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