Fong:RNA gel electrophoresis/Denaturing
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.
- 10x MOPS buffer, made using DEPC-treated water (see below)
- 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
- For 1% DEPC solution, add 2ml DEPC to 2L bottle and fill with nanopure H2O.
- Shake well and allow solution to incubate for at least 4 hours, or overnight if possible.
- Autoclave bottles on liquid cycle or for 10min at 120°C to remove traces of DEPC.
- Spray all glassware, casting tray, and gel box with RNAse Away and rinse well with DEPC treated water.
Denature RNA in buffer
- 5μL RNA sample (up to 30μg)
- 1μL 10x MOPS
- 3.5μL formaldehyde
- 10μL formamide
- Incubate at 60°C for 15-20min
Make Denaturing Gel (medium, 50mL gel)
- In a DEPC-H2O rinsed flask, combine
- 0.5g Agarose
- 4.2mL 10x MOPS
- 37.5mL DEPC-H2O
- 3.7μL formaldehyde
- Microwave for ~1.5 min until melted, cool to ~60°C
- 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.
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