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1. 32-P cDNA Synthesis
(Use silanized glassware and Eppendorf tubes throughout)
100 ul (1 mCi) aqueous 32P dCTP (10 mCi/ml, 800 or 400 Ci/mM)
500 mM tris 8.3
20 1OX CDNA buffer 60 mM MgCl2
600 mM NaCl
10 2OmM DATP
10 2OmM DGTP from P-L Biochemicals; stored at -700C
10 2OmM DTTP
3.3 2mM DCTP (it is important that the final concentration of dCTP be about 36uM to more full length material)
" 1 mg/ml oligo dT
4 1 M DTT
20 1 mg/ml Actinomycin D
2.5 - 5.0 ug poly A+ MRNA
Bring to 200 ul with dH20
10-20 un reverse transcriptase per ug RNA Incubate 420C, 2 hrs.
2. Base hydrolysis of RNA
Bring to 0.1 M NAOH
Heat 700 for 20 min to hydrolyze RNA Cool to rt, neutralize with HCl
Add NaAc (6.5) to 0.2 M
SDS to 0.1% (for column exclusion)
3. G-50 exclusion
Exclude CDNA on 2 ml G-50 F column in Pasteur pipette plugged
with silanized glass wool. Take 0.1 ml fractions.
Assess yield. Should be 40-80% of RNA
Column buffer: 100 mM NaCl
50 mM tris 7.5-8.0
1 mM EDTA
0.2% SDS
Combine exluded fractions (7-12)
Bring to 0.2 M NaCl by concentration with sec-butanol
62
+ chloroform extraction; no NaCl added
0-01 M MgCl2
Add 50 ug TRNA as carrier, 2.5 volumes EtOH, freeze on dry ice, cfg 30 min Sorvall SS-34 rotor, 15K. (The adapters designed for two standard test tubes fit Eppendorf tubes.)
3xlo5 cpm/m,
20 ml 10 filters
probes 5xl05 cpm/
1.2-1.6xl08 cpm/ug
4. RNA Reactions
Dry ETOH ppt. Resuspend in 2 ul TE (5mM tris, 1 mM EDTA) Typical reaction would be:
1.5 ul 32P cDNA
5.5 RNA for absorption
1.0 4M PB (phosphate buffer)
.1 ul 20% SDS
.1 100 mM EDTA
8.0 ul in 20 ul capillary
Heat at 90-1000C for 30 seconds
Incubate to Cot 1000-2000 at 680C
(for 4-2O hr reaction to avoid CDNA breakdown.)
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