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 l distilled deionized water (ddH2O).

6. Add 2 l of 5X Annealing Buffer (see Hint #2).

7. Incubate for 90 min at 60C (see Hint #3).

8. Microcentrifuge briefly every 30 min to avoid drying of the RNA.

9. Add 23 l PE mix and 10 Units Reverse Transcriptase (see Hint #4).

10. Mix carefully, microcentrifuge briefly, and mix again.

11. Incubate for 1 hour at 37C.

12. Add 300 l ice-cold 100% Ethanol.

13. Incubate in an ice bath for 15 min.

14. Microcentrifuge the pellet RNA/DNA for 10 min at 4C.

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 l 0.1 M NaOH.

19. Add 4 l 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 37C)
0.33 mM dATP
0.33 mM dGTP
100 g/ml Actinomycin D
0.33 mM dTTP
Store -20C 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


70% (v/v) Ethanol


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