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:
Clip 5mm mouse tail with a clean razor blade, place in 1.5 Ll tube, and store at -20 ̊C until further processing.
Add 100 μL of 1xMGB
Heat at 95 ̊C for 10 minutes
Add 110 μL of Proteinase K mastermix (10 parts 1x MGB, 1 part 20 mg/mL Proteinase K)
Incubate at 55 ̊C for 30 minutes
Vortex vigorously for 1 second
Repeat steps 5-6 two more times
Spin tubes at 14,000 x g for 5 seconds
Heat at 95 ̊C for 10 minutes
Spin at 14,000 x g for 5 minutes. Store at -20 ̊C until use. Use 1 μL per 50 μL PCR reaction.