Fong:RNA gel electrophoresis/Denaturing

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Overview

Gel electrophoresis separates RNA in essentially the same way as DNA, but since RNA often folds into a native conformation, it is necessary to denature the RNA strands if separation by size is needed. The RNA is denatured in this protocol by incubating with fomaldehyde at 60°C.

As with all RNA work, great care must be taken to avoid any contamination with RNAse.

Materials

• Agarose
• DEPC
• 10x MOPS buffer, made using DEPC-treated water (see below)
• Formaldehyde
• Formamide
• RNAse Away spray
• RNAse free filter pipette tips
• Gel casting tray and comb
• Gel box
• Electrophoresis power supply

Procedure

Treating water with DEPC to remove RNAse

1. For 1% DEPC solution, add 2ml DEPC to 2L bottle and fill with nanopure H₂O.
2. Shake well and allow solution to incubate for at least 4 hours, or overnight if possible.
3. Autoclave bottles on liquid cycle or for 10min at 120°C to remove traces of DEPC.
4. Spray all glassware, casting tray, and gel box with RNAse Away and rinse well with DEPC treated water.

Denature RNA in buffer

1. Combine:
   • 5μL RNA sample (up to 30μg)
   • 1μL 10x MOPS
   • 3.5μL formaldehyde
   • 10μL formamide
2. Incubate at 60°C for 15-20min

Make Denaturing Gel (medium, 50mL gel)

1. In a DEPC-H₂O rinsed flask, combine
   • 0.5g Agarose
   • 4.2mL 10x MOPS
   • 37.5mL DEPC-H₂O
   • 3.7μL formaldehyde
2. Microwave for ~1.5 min until melted, cool to ~60°C
3. Add 3.5μL ethidium bromide, and pour into casting tray.

Loading and running gel

5μL of the orange formaldehyde loading dye works for 1-30μg RNA. Modify amounts of RNA sample and dye to get strong enough bands, depending on the sample type and experiment requirements. Run the gel at 120V for 45-50min, or, for better resolution, 100V for 1hr.

Notes

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Contact

Deng
Chris

or instead, discuss this protocol.