|
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 Supplies Tips (责任编辑：泉水) |
RNA Primer Extension
时间:2005-07-18 00:00来源:Scienceboard.net 作者:admin 点击:
283次
顶一下
(0)
0%
踩一下
(0)
0%
------分隔线----------------------------
- 发表评论
-
- 最新评论 进入详细评论页>>
