Knight:RNA electrophoresis/Denaturing

Overview

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.

Materials

Reagents

• 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
• Glyoxal
  • 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

Equipment

• Electrophoresis apparatus

Procedure

Prepare RNase free water

1. Add DEPC to final concentration of 0.1% to H₂O
2. Incubate 1hr at 37°C.
3. Autoclave for 15 mins at 15 psi.

Prepare BPTE electrophoresis buffer

1. 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
2. Treat the solution with final concentration of 0.1% DEPC for 1 hour at 37°C
3. Autoclave.

Prepare glyoxal reaction mixture

1. 6mL DMSO
2. 2mL deionized glyoxal
3. 1.2mL of 10X BPTE electrophoresis buffer
4. 0.6mL of 80% glycerol

Divide into small aliquots and store at -70°C.

References

1. Molecular Cloning: Separation of RNA According to Size: Electrophoresis of Glyoxylated RNA through Agarose Gels