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RNA Primer Extension
Posted on Monday, October 20, 2003
Description
RNA Primer Extension
Procedure
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 ìl distilled deionized water (ddH2O).
6. Add 2 ìl 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 ìl 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 ìl 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 ìl 0.1 M NaOH.
19. Add 4 ìl 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
Recipes
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
0.3 M Sodium Acetate
0.1 M NaOH
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 ìg/ml Actinomycin D 0.33 mM dTTP Store -20°C in the dark. 10 mM MgCl2 0.33 mM dCTP
Annealing Buffer (5X) 1.25 M KCl 10 mM Tris-HCl, pH 7.9 1 mM EDTA
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