I am having problems with DNA extraction of a tail sample. Here is the procedure I've been using.
300 uL of cell lysis solution, then I use a blue microtube pestle to break up the tail. I add 1.5 uL of proteinase K at 20 mg/mL overnight on 55 degrees. I spin down the pellet the next day, then pour off the lysate fluid.
I add 1.5 uL RNase A at 4mg/mL invert several times and then incubate for 15-20 minutes.
I add 300 uL of protein precipitate solution and then vortex the solution for about 20 seconds. I centrifuge the solution at max speed, then put it on ice. I then vortex it again, centrifuge again, then pour off the lysate.
The lysate supernant goes into a new microtube with 300uL 100% isopropyl, mixed and then centrifuged for 2 minutes. I pour off the isopropyl and then add 70% Ethanol. I centrifuge and then invert the solution. Then pour the ethanol out.
FInally i add 50 uL of DNA hydration solution.
I use a nanodrop to determine the concentrations, and i get mostly low concentrations (3-6 ng/uL). The numbers for the 260/280 are mostly 1.50-1.85 with only a few going below 1.5
May I ask what do you need this DNA for? Genotyping? If yes, consider skip purification. In my experience it works just fine without it
Hi in my old protocol I perform the same passage with some variation.
I put directly the RNase in the lysis buffer and the perform the incubation ON. The day after spin directly and procede with the the extraction, with isopropanol or with phenol: clorophorm. I do not use vortex, or put on ice the sample during the extraction. If there is some problem to better dissolve the DNA in TE you can put the tube at 37°C ON or at 55°C fot 2-3h. Check if the problem is in the lysis buffer or at last step (DNA not dissolved well), if you have some samples try to put them at 37°C or 55°C, control not only with nanodrop but also with agarose gel.
Hi, in this step: "The lysate supernant goes into a new microtube with 300uL 100% isopropyl, ..." you can add between 30-50 uL from 3-7.5 M ammonium acetate, mix by inverting the tube and incubate for 30 min at - 20C. After incubation centrifuge on 12 000 rpm at + 4 C for 15-20 min. After that wash with 70% ethanol, repeat centrifuge tubes on 12 000 rpm at + 4 C for 7 min. and continue with your step.
You want to make sure you grind the sample well before adding lysis buffer.
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