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To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents and change gloves regularly while working. Treat water and solutions with DEPC (Diethyl Pyrocarbonate) to inactive RNases and use solutions prepared with RNase-free water and equipment. For more information about precautions when working with RNA, see Reference Pages under Working with RNA.
1. Precipitate the RNA with 5' end-labeled primer in 0.3 M Sodium Acetate with 2.5 volumes of 100% Ethanol (See Hint #1).
2. Mix well by inversion, microcentrifuge to pellet the RNA and discard the supernatant.
3. To the pellet add an equal volume of 70% Ethanol, mix well by inversion, microcentrifuge to pellet the RNA.
4. Decant the supernatant, dry the pellet by inverting the tubes over a paper towel and allow to air dry for a couple of minutes.
5. Resuspend the RNA in 8 μl distilled deionized water (ddH2O).
6. Add 2 μl of 5X Annealing Buffer (see Hint #2).
7. Incubate for 90 min at 60°C (see Hint #3).
8. Microcentrifuge briefly every 30 min to avoid drying of the RNA.
9. Add 23 μl PE mix and 10 Units Reverse Transcriptase (see Hint #4).
10. Mix carefully, microcentrifuge briefly, and mix again.
11. Incubate for 1 hour at 37°C.
12. Add 300 μl ice-cold 100% Ethanol.
13. Incubate in an ice bath for 15 min.
14. Microcentrifuge the pellet RNA/DNA for 10 min at 4°C.
15. Discard the supernatant and to the pellet add an equal volume of 70% Ethanol.
16. To the pellet add an equal volume of 70% Ethanol, mix well by inversion, microcentrifuge to pellet the RNA.
17. Decant the supernatant, dry the pellet by inverting the tubes over a paper towel and allow to air dry for a couple of minutes.
18. Redissolve the pellet in 2 μl 0.1 M NaOH.
19. Add 4 μl Formamide Dyes.
20. Place microcentrifuge tubes in boiling water bath for 2 min.
21. Incubate in an ice bath for 15 min.
22. Load sample on sequencing gel for electrophoresis (see Gel Sequencing Protocol).
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| 0.3 M Sodium Acetate |
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| Formamide Loading Buffer |
| 10 mM EDTA
1 mg/ml Bromophenol Blue
80% (v/v) Formamide (CAUTION See Hint #5)
1 mg/ml Xylene Cyanol FF
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| 0.1 M NaOH |
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| PE Mix |
| 5 mM DTT
20 mM Tris, pH 8.7 (at room temp; pH 8.3 at 37°C)
0.33 mM dATP
0.33 mM dGTP
100 μg/ml Actinomycin D
0.33 mM dTTP
Store -20°C in the dark.
10 mM MgCl2
0.33 mM dCTP
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| Annealing Buffer (5X) |
| 1.25 M KCl
10 mM Tris-HCl, pH 7.9
1 mM EDTA
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| 70% (v/v) Ethanol |
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DTT
Xylene Cyanol FF
Potassium Chloride
Bromophenol Blue
Sodium Chloride
EDTA
Sodium Acetate
Primer
Actinomycin D
dCTP
dTTP
dGTP
dATP
Ethanol
Reverse Transcriptase
RNA Carrier, Yeast
Magnesium Chloride
Oligonucleotide
Tris-HCl
Formamide
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1. If working with low amounts of RNA add some Yeast RNA Carrier (approximately 10 μg).
2. Double the volumes of dd H2O and annealing buffer if using more than 20 μg RNA (i.e. 16 μl ddH2O and 4 μl 5X Annealing Buffer).
3. The annealing temperature depends on the GC content of the RNA sample. The temperature of 60°C is acceptable for 20 to 30 base pairs with 50 to 70% GC content.
4. Double the amounts added if annealing amounts were doubled (i.e. 46 μl PE mix and 20 Units Reverse Transcriptase).
5. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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