< Knight:RNA electrophoresis
Electrophoresis permits assessment of RNA by size and amount. In general, electrophoresis of RNA is done as a step prior to Northern analysis. However, this protocol is for visualizing the RNA in the gel.
- 10X BPTE electrophoresis buffer (The final pH of this 10x buffer is ~6.5.)
- 100 mM PIPES
- 300 mM Bis-Tris
- 10 mM EDTA
- HPLC grade or better DMSO
- Commercially available stock solutions of glyoxal contain both hydrated forms of glyoxal and oxidation products that can degrade RNA. These must be removed.
- Glyoxal reaction mixture (divide into small aliquots and store at -70°C)
- 6mL DMSO
- 2mL deionized glyoxal
- 1.2mL of 10X BPTE electrophoresis buffer
- 0.6mL of 80% glycerol
- RNA gel loading buffer
- RNAse free water (make lots for rinsing glassware and electrophoresis chambers)
- Add DEPC to final concentration of 0.1%.
- Incubate 1hr at 37°C.
- Autoclave for 15 mins at 15 psi.
- SYBR Gold
Make up BPTE electrophoresis buffer
- Prepare by adding the following to 90 ml of distilled H2O
- 3 g of PIPES (free acid)
- 6 g of Bis-Tris (free base)
- 2 ml of 0.5 M EDTA
- Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C