Transgenic DNA Isolation From Mouse Tails
1. Chop up 1 cm long piece of <2 week old mice tail with two razor blades on new Petri dish. Transfer all to an Eppendorf tube.
2. Add 700 ml of lysis buffer. Place on rocker platform. Incubate 55°C o/n.
Lysis Buffer is:
50 mM Tris-HCl, pH 8.0 35 ml 1M Tris
100 mM EDTA 140 ml 0.5M EDTA
0.5 % SDS 35 ml 10% SDS
500 mg/ml proteinase K 35 ml 10mg/ml proteinase K
455 ml H2O
Make 500ml aliquots of 10mg/ml stock of PK in H2O. Store at -20. Thaw once only.
3. Add 700 ml phenol. Place on rocker platform to mix, RT, 3.
4. Spin max speed, 3 in microfuge.
3b. Transfer all of supernatant and interface into a new tube containing 700 ml phenol.
Place on rocker platform to mix, RT, 3.
4b. Spin max speed, 3 in microfuge.
5. Transfer all of supernatant and interface into a new tube containing 700 ml phenol:CHCl3. Place on rocker platform to mix, RT, 3.
6. Spin max. speed, 3 in microfuge.
7. Transfer all of supernatant and interface into a new tube containing 700 ml CHCl3:Isoamyl alcohol. Place on rocker platform to mix, RT, 3.
8. Spin max. speed, 3 in microfuge.
9. Transfer supernatant (Interface should now be clear) to a Sarsteadt 15ml tube.
Add 350 ml 7.5M NH4oAc (1/2 Aq volume)
Add 2.6 ml ethanol.
Mix. DNA should form large fluffy white precipitate immediately. If so pipette the DNA directly into a microfuge tube containing 1 ml 70% ethanol. Go to step 10.
Otherwise incubate in dry ice, 30 min. Spin in J13.1, 8500 rpm, 30 min., 4°C.
10. Wash DNA in 1 ml 70% ethanol, transfer to microfuge tube. Vortex to remove residual SDS. Spin max. speed, 5 min., RT.
11. Rewash in 1ml 70% ethanol. Spin max. speed, 5 min., RT.
12. Speedvac dry, 10 min. No heat.
13. Resuspend in 200-300 ml TE. Rocker, 55°C, overnight.
14. Quant 1 ml with standards.
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