Mouse Tail Genomic DNA Isolation Protocol(1) ( ...)

Mouse Tail Genomic DNA Isolation Protocol(1)

Note: Genomic DNA is fragile. High molecular weight DNA is sheared easily by mechanical forces. Use suitable large-bore pipet tips or equipment when pipetting genomic DNA. Do not vortex solutions containing genomic DNA.

Place a 1 cm tail sample into a 1.5 ml microcentrifuge tube; this may be stored at 20C. To minimize possible cross-contamination, do not mince the sample. Add 700 l Tail Buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% SDS) to the sample.
Add 35 l 10 mg/ml Proteinase K to the sample and mix briefly.
Incubate at 5560C overnight with mixing. This step should result in the complete solubilization of the tail fragment. In the case of incomplete digestion, more Proteinase K can be added and the samples incubated for several more hours.
Add 20 l 10 mg/ml RNase A (DNase-free) to the sample. Mix briefly and incubate at 37C for 12 hours.
Transfer entire solution to a pre-spun (1500 x g for 12 minutes) PLG 2 ml Heavy tube.
Add 0.5 ml Phenol-Chloroform-Isoamyl Alcohol (PCI, 25:24:1) to the sample in the PLG 2 ml tube and mix well by repeated inversion. Do not vortex.
Centrifuge at full speed (12,000 x g or greater) for 5 minutes in a microcentrifuge, then carefully transfer the resultant aqueous phase to a fresh pre-spun PLG 2 ml Heavy tube.
Add 0.5 ml Chloroform-Isoamyl Alcohol (CI, 24:1) to the sample in the PLG 2 ml tube and mix well by repeated inversion. Do not vortex.
Centrifuge at full speed (12,000 x g or greater) for 5 minutes in a microcentrifuge, the
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( ...)Mouse Tail Genomic DNA Isolation Protocol(1)