Fong:RNA gel electrophoresis/Denaturing

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

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:
 * 2) * 5μL RNA sample (up to 30μg)
 * 3) * 1μL 10x MOPS
 * 4) * 3.5μL formaldehyde
 * 5) * 10μL formamide
 * 6) Incubate at 60°C for 15-20min

Make Denaturing Gel (medium, 50mL gel)

 * 1) In a DEPC-H2O rinsed flask, combine
 * 2) * 0.5g Agarose
 * 3) * 4.2mL 10x MOPS
 * 4) * 37.5mL DEPC-H2O
 * 5) * 3.7μL formaldehyde
 * 6) Microwave for ~1.5 min until melted, cool to ~60°C
 * 7) 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.

Contact
Deng

Chris

or instead, discuss this protocol.