Mouse Tail DNA Extraction
a la Lester Lab January 2004
1. Remove the last 3-5 millimeters of the mouse tail for genotyping. Any more than 5 mm of tissue will not digest well; if possible, use a biopsy no bigger than 3mm. Really. I mean it. Very, very small.
2. Add 250 ml of 50 mM NaOH to each tail. Incubate the tissue at 95° C for approximately 45 minutes (use a heat block). Beware: tubes heated in this manner can open and spew near-boiling NaOH if disturbed. Wear gloves and glasses if this is a problem.
3. At the end of the 95° C incubation, a tube carefully held closed and vortexed should contain only hair and a small bit of cartilage. The soft tissue should completely dissolve. Once at least one tail disintegrates when vortexed, turn off the heat block and leave the tubes in a place until they cool a bit (to roughly 50-70° C). Once the block and tails have cooled, vortex each tube to fully digest the tissue.
4. Neutralize each sample by adding 250 ml of 0.5M Tris-HCL pH 5.5 to the digest. Mix well. Check the pH of a few samples with paper – it should not exceed 8.0.
5. Centrifuge 1-2 minutes at high speed to sediment debris.
6. Use 3 ml of the sample for PCR. Because so little tail tissue is used in this protocol, I tend to add 2-3 extra cycles to PCR programs developed for PK-digested/EtOH-precipitated DNA.
Mouse Toe DNA Extraction
July 2012
1. Mice should be between P5 and P12 for this procedure. See Schaefer et al, Lab Animals 44:7-13 (2010) for reference. Cut toes at second phlange of digit and place each into one well of a thin-walled 96 well PCR plate.
2. Centrifuge at 4100 rpm for 10 minutes.
3. Add 50 ml of 50 mM NaOH to each tail. Incubate the tissue at 95° C for approximately 30 minutes (use PCR machine).
4. At the end of the 95° C incubation, vortex the plate for 1-2 min to break up any pieces of tissue.
5. Centrifuge at 4100 rpm for 10 minutes.
6. Neutralize each sample by adding 50 ml of 0.5M Tris-HCl pH 5.5 to the digest. Vortex to mix.
7. Centrifuge at 4100 rpm for 10 minutes before drawing supernatant for PCR.
8. Use 3 ml of sample for PCR. Because so little tissue is used in this protocol, I tend to add 2-3 extra cycles to PCR programs developed for PK-digested/EtOH-precipitated DNA.