DNA purification for PCR
Protocol for isolating DNA from embryo yolk sacs, or toes, or 2 mm tail for PCR.
1. Dissect embryos and place each yolk sac in a microfuge tube (can be frozen at -20°C prior to extraction). Original method cited for mouse toes, or 2 mm tail,
A) 25 mM NaOH / 0.2 mM EDTA. The pH is about 12.
B) 40 mM Tris HCl (not Tris base). The pH is about 5. Don’t adjust the pH.
For yolk sac/placenta/tail/toes
2. Add 40 µl (100 µl for tail/toe) 25 mM NaOH / 0.2 mM EDTA.
3. Place at 95°C for 20 minutes (in a thermal cycler)
4. Add 40 µl (100 µl for tail/toe) 40 mM Tris HCl. 5. Vortex. The DNA is now in a 20 mM Tris/0.1 mM EDTA at pH 8.1 with about 12.5 mM Na++ ions. That’s pretty close to the 10 mM Tris/ 0.1 mM EDTA / pH 8 solution used to store DNA.
6. Use 1 µl for PCR
7. (May need to microfuge for 6 minutes and transfer supernatant to new tube for storage (at 4 or -20 degrees C).