Knight:RNA electrophoresis/Denaturing

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Revision as of 17:31, 5 December 2006

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

• 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.
• 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 size marker
• RNA gel loading buffer

• SYBR Gold

Equipment

• 37 °C incubator
• 55 °C water bath
• Ice water bath
• 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 10X buffer 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.

Dilute 10X buffer 10-fold with RNase free H₂O.

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.

Denature RNA samples

1. Mix 10 μL glyoxal reaction mixture with 1-2 μL RNA (up to 10 μg).
2. Also mix 10 μL glyoxal reaction mixture with RNA size marker.
3. Incubate RNA samples at 55°C for 1 hr.
4. Chill RNA samples for 10 mins in ice water.
5. Centrifuge 5 seconds to collect liquid at the bottom of the tubes.

Cast gel

Do this step during 1 hr denaturation of samples.

1. Cast 1.5% agarose gel in 1X BPTE electrophoresis buffer.
2. Use a comb with at least 4 extra lanes for size markers and running dyes.
3. Place gel in chamber.
4. Cover with 1X BPTE electrophoresis buffer to cover gel to a depth of 1mm.

Notes

References

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