This was the protocol I used when I genotyped mice. It’s not quite as dirty as running a PCR off a proteinase K tail digestion straight, but it happily still manages to avoid the nasty phenol:chloroform:isoamyl alcohol use.
Necessary stock solutions
- Proteinase K
- Tail lysis buffer (some low-pH buffer like Tris with some sort of detergent in it like SDS)
- 70% ethanol (it’s good for what ails ya and every lab should have access to an abundant supply of ethanol)
- Isopropanol
- TE for reconstitution or water in a pinch
Digest the tail
I keep proteinase K in a freezer-proof stock solution that includes glycerol so it stays in the -20 but doesn’t succumb to the effects of freeze-thawing. For every tail you have, you’ll want one unit of 500ul of tail lysis buffer that includes 100ug of proteinase K.
In 1.5ml eppendorf tubes, put a piece of tail, the lysis buffer, and the proteinase K. (Often it’s a good idea to take a little bit of the tail and set it aside in case something goes wrong with the extraction. It saves having to retail the mice.) Put the tubes (labeled) on a hot plate at 55 degrees C overnight. You can also put them in a water bath.
Get rid of the gunk
The next morning, vortex the samples to loosen the hairs and tidbits from the DNA. Don’t be too zealous about it as you’ll end up breaking the DNA. In fact, if you aren’t sure how fast to vortex it, don’t vortex it at all. Just invert the tube a couple times. If the tails aren’t yet fully digested, let them sit at 55 degrees for a while longer and consider getting some new proteinase K for next time.
Spin down the samples for 10 minutes (5 minutes is fine) at 13.2krpm (or whatever the fastest setting on the centrifuge is). The DNA you want to keep is now in the clear liquid at the top.
Transfer the liquid to new (labeled) tubes.
Precipitate the DNA
To these new tubes you want to add 500ul (or again the same volume as the DNA in buffer) of isopropanol (also known as 2-propanol). Invert the tube 15 times and if you’re lucky (or skilled) you’ll see a very small white thread-looking thing (knotted) floating in the buffer. If you don’t see it don’t throw the tube away. It might still be in there.
Spin down the tubes again for 5 minutes to get the now-visible DNA in the bottom of the tube.
Clean the DNA
Once the DNA is pelleted in the bottom of the tube, either use a pipetman (Gilson is my favorite) to remove the supernatant. Else, if you’re feeling courageous or if you’re pressed for time, just invert the tube over a waste beaker. The waste can go down the drain (if the isopropanol smells, run some water). Before turning the tube right side up again, touch it briefly to a fresh area on a kimwipe. This decants that last little droplet and helps the purity of the reconstituted DNA as well as helps the tube dry faster.
Add 1ml of 70% ethanol. Invert the tube a couple times. If the pellet moves, spin it down again (30 seconds will do). Repeat this step to rinse the pellet 3 times in 70% ethanol.
Dry and Reconstitute
After the third ethanol wash, remove the ethanol and let the tube dry on its side. I put the tubes under a fresh kimwipe to prevent dust from getting in there and I write something like “drying tubes, do not touch” on a paper towel and point to the tubes.
After an hour has gone by (or overnight) or however long it takes for the DNA to dry and then some, add 50ul of TE. If the pellet is small add 20ul. Remember: you can always add more TE but you can’t take it away (at least not after the pellet is redissolved).
Once again, leave the tubes at 55 degrees C overnight in the new TE buffer to allow the DNA to relax and, as my mother would have said, to groove with the buffer.
