Materials and Reagents

Notes:

1. It is crucial to keep all the materials and reagents RNase-free for this protocol. To this end, the reagents and materials are all RNase-free, and are purchased pre-made or ready-to-dissolve in order to simplify the procedure as much as possible. Disposable RNase-free centrifuge tubes are used to make reagents whenever applicable.

2. Multiple kits are available for DIG reagents. Items 19-23 are listed here for readers to choose from to suit the scale required.

   
   

1. Centrifuge tubes, RNase free (1.5 ml, 2 ml, 15 ml and 50 ml, as needed)

2. QIAzol Lysis Reagent (Qiagen, catalog number: 79306 )

3. Chloroform, molecular biology grade

4. Isopropanol, molecular biology grade

5. Ethanol, molecular biology grade

6. UltraPure DNase/RNase-Free Distilled Water (Invitrogen, catalog number: 10977-023 )

7. TEMED (National Diagnostics, catalog number: EC-503 )

8. Ammonium persulfate (National Diagnostics, catalog number: EC-504 )

9. Urea (National Diagnostics, catalog number: EC-605 )

10. AccuGel 29:1 (30%) (National Diagnostics, catalog number: EC-851 )

11. Sodium acetate, 3 M solution, pH 4.5 (National Diagnostics, catalog number: EC-905 )

12. Sodium acetate, 3 M solution, pH 5.2 (National Diagnostics, catalog number: EC-906 )

13. EDTA, 0.5 M, pH 8.0 (Thermo Fisher Scientific, catalog number: AM9260G )

14. Xylene cyanol, molecular biology grade

15. Bromophenol blue, molecular biology grade

16. Tris-HCl solution, 1 M, pH 9.0 (Sigma-Aldrich, catalog number: T2819-100ML )

17. Amersham Hybond-XL membrane (GE Life Sciences, catalog number: 45-001-148 )

18. Tris-broate-EDTA (TBE) buffer, 10x (Thermo Fisher Scientific, catalog number: AM9863 )

19. DIG RNA labeling Mix (Sigma-Aldrich, catalog number: 11277073910 )

20. DIG RNA labeling kit (Roche, catalog number: 11175025910 )

21. DIG system (Roche, catalog number: 12039672910 )

22. DIG Easy Hyb (Sigma-Aldrich, catalog number: 11603558001 )

23. DIG Wash and Block Buffer Set (Sigma-Aldrich, catalog number: 11585762001 )

24. Anti-Digoxigenin-AP, Fab fragments (Sigma-Aldrich, catalog number: 11093274910 )

25. CDP-Star (Sigma-Aldrich, catalog number: 11685627001 )

26. RNase Zap (Thermo Fisher, catalog number: AM9780 )

27. Growth medium for R. palustris (PM medium) (see Recipes)

28. Acid-urea gel (see Recipes)

29. Sodium acetate-EDTA running buffer (see Recipes)

30. Sodium acetate-EDTA loading buffer (see Recipes)

Equipment

Note: The working bench as well as electrophoresis, transfer and Northern blot systems should be cleaned with RNaseZap or equivalent to decontaminate RNase before use.

1. Zirconia/Silica Beads, 0.1 mm dia (Bio Spec Products Inc, manufacture number: 11079101z )

2. Mini-Beadbeater-24 (Bio Spec Products Inc, catalog number: 112011 )

3. Electrophoresis, transfer and Northern blot system that fits a gel 20 cm in height

4. Temperature-controlled tabletop centrifuge

5. PCR machine for temperature-controlled incubation or equivalent incubator

Software

1. ImageJ

Procedure