Knight:RNA electrophoresis/Denaturing

in progress! has not been tested

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
  • 95% deionized formamide
    • Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
  • 0.025% (w/v) bromophenol blue
  • 0.025% (w/v) xylene cyanol FF
  • 5 mM EDTA (pH 8.0)
  • 0.025% (w/v) SDS

• 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.

Prepare RNA gel loading buffer

1. Mix the following
   • 95% deionized formamide
     • Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20°C.
   • 0.025% (w/v) bromophenol blue
   • 0.025% (w/v) xylene cyanol FF
   • 5 mM EDTA (pH 8.0)
   • 0.025% (w/v) SDS

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.

Run gel

1. Add 1-2μL RNA gel loading buffer to glyoxylated RNA samples
2. Immediately load samples. Leave two outside lanes on each side empty.
3. Load RNA size markers on outside lanes of gel.
4. Electrophorese at 5V/cm.

Stain gel

1. Prepare fresh 1:10,000 dilution in RNase free water of SYBR gold.
2. Ensure pH is 7.0-8.5.
3. Pour into staining tray.
4. Place gel in plastic staining container.
5. Shield from light.
6. Agitate gently for 10-40 mins at room temperature.
7. Image with Knight:Gel imager. (Place a clear ruler next to gel to more accurately assess length.)

Notes

• Avoid RNase contamination

References

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