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.