|
Transgenic Core Facility
|
||||||||||
|
1) Chop up 1 cm long piece of mouse tail with two new 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 over night. 3) Add 700 ml of phenol. Shake tube to mix at room temperature (RT) for 3 minutes. 4) Spin at max speed for 3 minutes in microfuge. 5) Transfer all of supernatant and interface into a new tube containing 700 ml phenol. Shake tube to mix at RT for 3 minutes. 6) Spin at max speed for 3 minutes in microfuge. 7) Transfer all of supernatant and interface into a new tube containing 700 ml phenol:CHCl3(1:1 ratio). Shake tube to mix at RT for 3 minutes. 8) Spin at max speed for 3 minutes in microfuge. 9) Transfer all of supernatant and interface into a new tube containing 700 ml CHCl3:Isoamyl alcohol (24:1 ratio). Shake tube to mix at RT for 3 minutes. 10) Spin at max speed for 3 minutes in microfuge. 11) Transfer supernatant (Interface should now be clear) to a Sarsteadt 15ml tube. Add 1/10 volume of 3M NaOAc (pH 5.5) and 2 volume of 100% ethanol. Shake tube. DNA should form large fluffy white precipitate immediately. If so, spin for 10 minutes in microcentrifuge, otherwise incubate in dry ice for 30 minutes. Then spin. 12) Take out solution and wash DNA in 1 ml of 70% ethanol. Spin at max speed for 5 minutes at RT. 13) Take out solution and rewash DNA in 1 ml of 70% ethanol. Spin at max speed for 5 minutes at RT. 14) Air dry for few minutes. Do not over dry. 15) Resuspend in 200-300ml of TE (pH 8.0). Place on rocker platform at 55°C to resolve over night. 16) Quant 1 ml with standards.
Lysis buffer 50 mM Tris-HCl, pH 8.0 -- 1M Tris 35 ml 100 mM EDTA -- 0.5M EDTA 140 ml 0.5 % SDS -- 10% SDS 35 ml 500 mg/ml proteinase K -- 10mg/ml proteinaseK 35 ml H2O 455 ml
TE (pH 8.0)
10mM Tris (pH8.0) 1mM EDTA (pH8.0)
|
|||||||||
