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The Laboratory of Andrew Murray at Harvard University
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A. Alkali Treatment of RNA (see Hint #2 and #3)
1. Take 2 to 5 μg of RNA (see Hint #4) and dilute to 18 μl using DEPC-treated ddH2O. Use approximately 0.5 μg RNA per filter of cDNA library to be screened.
2. To the RNA, add 2 μl of 1 M NaOH, mix well.
3. Incubate the solution on ice for 10 to 15 min.
4. Add 2 μl of 3 M RNAse-free Sodium Acetate and 60 μl of 96% Ethanol.
5. Incubate on ice for 10 min.
6. Microcentrifuge the solution at maximum speed and carefully aspirate the supernatant (see Hint #5).
7. Wash the RNA pellet with 50 μl of 80% Ethanol.
8. Microcentrifuge the solution at maximum speed for 10 min and carefully aspirate the supernatant (see Hint #5).
B. 5- Endlabeling of RNA Probe by Polynucleoide Kinase
1. Redissolve the RNA pellet in 20 μl RNAse-free Tris Buffer.
2. Add 0.1 volume of final reaction volume of 10X T4 Polynucleotide Kinase Buffer. Use recipe given or buffer sent by manufacturer with enzyme.
3. Add approximately 125 μCi of radiolabeled ATP (see Hint #1).
4. Add 12 Units of T4 polynucleotide Kinase (0.5 μl).
5. Incubate for 30 min at 37°C.
6. Add RNase-free EDTA to a final concentration of 20 mM to stop the kinase reaction.
7. Remove a 1 μl aliquot of the solution and check radiolabel incorporation into the RNA by CTAB precipitation (see CTAB precipitation of RNA protocol) followed by scintillation counting.
8. To the remainder of the Kinase Reaction solution add 100 μl 100% Ethanol and mix well.
9. Apply solution to a 1 to 2 ml Sephadex G50 (fine) column packed in a Pasteur pipette and collect the first radiolabeled peak (use a survey meter to detect the peak) (see protocol on preparing Sephadex G50 Columns). Alternatively, use a commercial spin column for RNA purification.
10. Resuspend RNA in the appropriate buffer for probing cDNA library. Specific activity should be 1 X 107 cpm per μg RNA (or better).
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| 96% (v/v) Ethanol |
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| 3 M RNAse-free Sodium Acetate |
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| 1 M NaOH |
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| 125 μCi γ-[32P] ATP |
| CAUTION! see Hint #1
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| 0.5 M RNAse-free EDTA, pH 8.0 |
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| 24 Units/μl T4 Polynucleotide Kinase |
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| T4 Polynucleotide Kinase Buffer (10X) |
| 1 mM EDTA, pH 8.0
50 mM DTT
0.5 M Tris-HCl, pH 6.6
100 mM MgCl2
1 mM Spermidine HCl
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| RNase-Free Tris Buffer |
| 10 mM Tris-HCl, pH 8.3
10 mM MgCl2
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| 80% (v/v) Ethanol |
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Tris-HCl
Oligonucleotide
Diethyl Pyrocarbonate (DEPC)
I3-[32P]-ATP
Ethanol
Sodium Hydroxide
EDTA
Spermidine-HCl
Sodium Acetate, RNase-free
DTT
Polynucleotide Kinase, T4
Magnesium Chloride
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. To minimize degradation of RNA by RNAses, wear gloves when handling samples and reagents and change gloves regularly while working. Use sterile plasticware or glassware that has been baked at 150°C in dry heat for at least 2 hr. Autoclaving solutions and vessels is not sufficient to remove all RNase activity. DEPC treat water to inactive RNAses and use buffers prepared with RNase-free equipment and water. Do not use spatulas to measure out chemicals used for RNAse-free solutions. Reserve RNAse-free solutions, reagents, and consumables, such as pipette tips, only for RNA work.
3. Alkali hydrolysis of RNA cleaves the RNA, producing 5'-OH groups that are substrates for phosphorylation by the kinase.
4. Normally the RNA is poly A+.
5. Be careful not to aspirate the RNA (pellet which will be very hard to see) with the supernatant.
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