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2. Base Hydrolysis and Sephadex Exclusion Add equal volume of 0.2 M NAOH
Incubate 20 min., 700C
Cool on ice, netralize with HCl Add Tris (pH 8.0) to 0.1 M, SDS to 0.1%, warm to room temp. Exclude from G-50 F Sephadex
(2 ml Pasteur pipette column 0.1* ml fractions)
In column buffer 0.1 M NaCl
.05 M Tris 7.5
.001 M EDTA
.02 t SDS
3. Ethanol Precipitation
Pool the excluded peak of the Sephadex column
Bring to .2 M NaCl -01 M MgCl2
PPT in 2 volumes ETOK, freeze on dry ice
Spin in ultracentrifuge (SW50.1, 1 hr, 30K rpm)
4. Hybridization
Resuspend in 4.5 ul 0.1 mM EDTA
Add 6 ul 2 mg/ml Poly A+ MRNA, mix
Then add 1.5 ul 4.0 M Phosphate buffer (PB)
0.1 ul 20% SDS approximate 0.1 ul 100 mM EDTA
Take up mixture in 20 ul silonized capillary, heat seal ends
Heat to 900C for 30 seconds to disaggregate
Incubate 650C for 12-20 hrs (approximate termination for most complex mRNAs)
5. Hydroxvanetite Selection
(t RNA DNase treated)
Break ends of capillary with a triangular file (liver glycogen Sigma + NaO4 for Ppt at isopropanol for 2 hrs at 650C.
Dilute into 1 ml of 0.12 M PB 3, 0.1% SDS (Tom recommends'.48 M 600, 10% HCl 600)
Run over a 1 ml hydroxyapetite column (water jacketed) at 600C. It should take 20-30 sec. to come through. Take care when making column not to pack too tight and avoid generating holes.
Rinse with 1 ml aliquots of 0.12 M PB, 0.1% SDS (about seven)
Raise column temperature to 980C
Stir column and rinse through 5 fractions of PB (to measure hybridized fraction)
Count all fractions to determine percent hybridization (should be 90-95% for closely related materials)
Pool excluded peak (the best three fractions)
(BPB will comigrate with salt)
Concentrate with sec-Butanol to 0.2 ml
Sec butanol
Get rid of SDS as well
1 M .2M Na isopronal dry 151
Exclude over G-50 F Sephadex as before (to remove phosphate buffer)
Precipitate and spin in the ultracentrifuge as before.
6. Second Strand Synthesis Resuspend in 0.1 mM EDTA
Make 100 mM Hepes 6.9
12 mM MgCl2
100 mm Kci
10 mM DTT
1 mM each of dNTP's
DNA polymerase 1, 30 units/ug CDNA or
Klenau fragment it if to it
Incubate 4 hs., 150C with pol I or
30 min. room temperature with Klenow
7. Sl Cleavage
Add 4 volumes of lx Sl buffer .2 M NaCl
05 M NaAc (4.5)
:ooi m ZnSO4
and titrate material with Sl nulcease (look for abrupt shift in the single-strand length on a denaturing gel).
After Sl cleavage, bring to 5 mM EDTA, 0. 1% SDS and phenol extract.
Concentrate again with sec-butanol to 200 ul and run over G-50 F Sephadex as before.
PPT double-stranded material.
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