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RNA Purification
8) Adjust sup to 0.5% SDS
9) Add equal volume of phenol (equilibrated c TRIS to pH between 5 & 7) and vortex
10) Centrifuge at 3,000 RPM at room temp for 15 minutes, and Remove sup and extract with chloroform: 1SO-amyl alcohol (24:1)
11) cfg 3000, 10min, remove sup
12) Adjust solution to 0.3M NaAc pH 5.2 with 3M or 5M NaAc pH 5.2
13) Add 2.5 volumes ETOH - Vortex and place in -200C freezer OIN
14) Spin in sorvall at 7,000 RPM, 40C for 20 minutes
15) Wash pellet with 70% ETOH
16) Dry tubes with cotton swabs, then speed vac briefly
17) Resuspend pellet in DDH20 - (600 ul total volume for 8 plates usually gives final concentration of 1-2 ug/ul)
Modification for work with fresh tumor tissue:
Tissue ground in motor and pestle in liquid N2 powder then resuspended in:
10 mM pH 7.4
150 mM NaCl
1.5 mM MgCl
4 mM Vanadyl ribonycleoside complex
Remainder of preparation as above
DNA Purification
1) Nuclear pellet ground with motor and pestle in liquid N2 only if frozen, and to nuclei from 150 mm3 plate add 3 ml of PK solution
2) Resuspend in PK solution:
Work Stock Dil
NaCl 0.15M 5M 1:33 1.5 ml
EDTA .1m 0.5M 1:5 10.0 ml
TRIS pH 8 1.OM .02M 1:50 1.0 ml
50 ml
3) Adjust to 1% SDS. Stock 20% SDS
4) Add proteinase K 50 ug/ml - incubate 370C 3-8 hours or 450C 3-4 hours
5) Phenol extract (equilibrated c TRIS to pH 8 containing 0.1% 8-OH-quinolone)
6) cfg 151 at 3000 rpm DNA spooled in ethanol
Add 2 volumes ethanol and spool on glass rod
7) Resuspend wrapped DNA in .01M TRIS pH 7.5
Rock gently OIN at RT
8) RNASE (1 mg/ml stock) in 50 ug/ml, 1 hr, RT
9) Proteinase K: 3-4 hr, 370C
Adiust salt to: .15M NaCl .01M EDTA
1% SDS
Add PK 50 ug/ml
10) Phenol extract, cfg Chloroform/lSO-amy (24:1)
11) Dialyse vs 0.01M TRIS pH 8.0 or ETOH ppt
11) Spectrum
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