PREPARATION OF GENOMIC DNA FOR GENOTYPING
Tail Lysis Buffer (100 mM Tris.Cl pH8.0, 5 mM EDTA, 0.2% SDS, 200 mM NaCl)
50 ml 1 M Tris.Cl pH8.0
5 ml 500 mM EDTA pH8.0
10 ml 10% SDS
20 ml 5 M NaCl
Add dH2O to 500 ml
Store at room temperature
- Obtain a piece of tail (about 5 mm long is enough), put into an Eppendorf tube
- For adult mice, anesthetize the mice before cutting the tail. For embryos, decapitate the embryos before cutting the tail.
- Add 0.5 ml Tail Lysis Buffer and 5-10 l of 20 mg/ml Proteinase K
- Shake at 50-55 C overnight
- Efficient digestion is critical. If the digestion is not complete, it will be difficult to get a compact DNA pellet in later steps. Robust shaking greatly improves the digestion efficiency. To maximize the shaking, place the Eppendorf tubes flat instead of upright on a shaker. It may be helpful to use Eppendorf clamps to prevent the tubes from poping open during the shaking.
- Microcentrifuge at top speed for 10 min to pellet undigested hair, transfer the supernatant to a new Eppendorf tube. (Omit this step for embryonic tails)
- Add 0.5 ml isopropanol. Invert tubes a few times.
- Microcentrifuge at top speed for 10 min at 4 C.
- Pour off supernatant, wash pellet with 70% ethanol
- Dry the DNA pellet by Speed Vac (5 min, setting at Medium).
- Resuspend the DNA pellet in 100 μl of dH2O by pipetting up and down with a P200 pipetman.
- Use 2 μl of DNA for PCR (total PCR volume: 20 l).