DNA extraction from mouse tails
1. Cut tails, approximately 0.5 cm.
If you are not very experienced doing genotyping, then cut a 1 cm piece, divide into two halves. Use one half for DNA preparation, and save the other in separate tube in the freezer as backup. These backup tubes can be tossed if your results from the first half are clear-cut.
Store the mouse tail tips at -80°C freezer, before you have time to do extraction.
2. Thaw (briefly at 55°C) Proteinase K (20mg/ml) (see note 2) stock solution (calculate how much you will need for this experiment. Avoid freezing and thawing multiple times). Make sure the solution is clear.
Add 500 μl TNES buffer (Make sure the solution is clear. If you see white precipitates, make fresh solution) and 12.5 μl Proteinase K (20mg/ml) to each tube with tail in it.
Incubate at 55°C waterbath overnight (make sure tails are submerged in solution).
3. The next morning, check to see if digestion is complete. If you still see residual tissues, thoroughly mix the tubes and digest for another few hours. If digestion complete, centrifuge the tubes shortly to spin down the drops on the caps.
4. Add 150 μl 6M saturated NaCl (see note 3) into each tube. Vigorously shake for at least 40 seconds. Longer the better.
Spin down at full speed for 5 minutes at room temperature to pellet proteins and junk.
5. Remove about 500 μl supernatant and transfer into fresh tube. Careful: don’t take any pellet with you!
Add 500 μl 100% EtOH and mix by inverting the tubes. You should be able to see white thread-like material at this step. **If you are working with multiple tubes, make sure you invert each tube immediately after Ethanol addition!!
6. Spin at full speed for 5 minutes at room temperature to pellet the DNA.
Remove supernatant. Do this very carefully – do not pour or vacuum. Use a blue tip to remove the bulk of solution but a tiny bit behind. Then spin briefly and remove residual supernatant using a yellow tip. During the process, monitor the presence of pellet in each tube.
7. Wash pellets with 500 μl 70% EtOH. Remove supernatant as described above. Make sure not to leave any supernatant behind but at the same time not to lose the pellet.
8. Air dry at room temperature for about 10 minutes.
9. Add 100 μl TE (pH 8.0), incubate at 65 °C for 20 minutes. Flip the bottom of tube to resuspend. Place at room temperature overnight or for a few hours to facilitate resuspension.
10. Check the genomic DNA you get on a small DNA gel, load about 2 μl sample / each well.
Expected yield: 0.5 – 1 ug/ul, 100 ul = 50 – 100 ug.
Note 1: TNES buffer For 50 ml:
50 mM Tris-HCl (pH7.4) 1 M Tris-HCl (pH7.4) —– 2.5 ml
100 mM EDTA (pH8.0) 0.5 M EDTA (pH8.0) —– 10 ml
400 mM NaCl 5 M NaCl —– 4 ml
0.5% SDS 10 % SDS —– 2.5 ml
dd H2O —– 31 ml
Note 2: Proteinase K (20mg/ml)
—– Proteinase K powder is shipped and stored at -20°C freezer.
—– Making stock solution: add 2.5 ml dd H2O to the bottle(50 mg/each bottle)
and mix by inverting, then aliquot into small amount vials, ( usually 50 μl) and store in the Proteinase K stock solution box in the -20°C freezer.