Fong:RNA gel electrophoresis/Denaturing

Overview

This protocol is under construction

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.

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.

Notes

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