Gitschier Protocol for Genomic DNA isolation from mice

10x Modified Gitschier Buffer (10x MGB)

223 mL Tris (1.5 M, pH 8.8)= 670 mM Tris

10.97 g Ammonium Sulfate =166 mM Ammonium Sulfate

3 g Magnesium Chloride= 65 mM Magnesium Chloride

Fill to 500 mL with Milli-Q H₂0, mix well and autoclave

1x MGB (for 10 mL)

8.85 mL ddH₂O

1 mL 10x MGB

100 μL 2-mercaptoethanol

50 μL Triton X-100

Vortex vigorously to make it soluble Triton X-100

Proteinase K mastermix

10 parts 1x MGB

1 part 20 mg/mL Proteinase K

Method:

• 1. Clip 5mm mouse tail with a clean razor blade, place in 1.5 Ll tube, and store at -20 ̊C until further processing.

  2. Add 100 μL of 1xMGB

  3. Heat at 95 ̊C for 10 minutes

  4. Add 110 μL of Proteinase K mastermix (10 parts 1x MGB, 1 part 20 mg/mL Proteinase K)

  5. Incubate at 55 ̊C for 30 minutes

  6. Vortex vigorously for 1 second

  7. Repeat steps 5-6 two more times

  8. Spin tubes at 14,000 x g for 5 seconds

  9. Heat at 95 ̊C for 10 minutes

  10. Spin at 14,000 x g for 5 minutes. Store at -20 ̊C until use. Use 1 μL per 50 μL PCR reaction.