Jacobs:Protocol RNA Agarose Gel

Overview

Protocol for preparing and running an RNA agarose gel useful for determining RNA quality.

Materials

• Agarose
• Formaldehyde
• 10X MOPS buffer
• RNAse free water
• Ethidium Bromide
• Glass bottle
• RNA Sample
• Microwave
• Agarose Gel Electrophoresis system
• Blue Juice dye
• DNA ladder
• Ice bucket
• Ice
• Serological pipets
• Pipet aid
• Pipetter
• Pipet tips
• RNAse free microfuge tube
• UV transilluminator
• Camera
• Ethidium bromide extractor

Procedure

1. In fume hood, add 1.95g agarose, 108.23ml RNAse free water, 13ml 10X MOPS in a 500 ml or larger glass bottle. (This makes 1.5% agarose gel solution)
2. Heat until solution is clear and boiling in the microwave. It will take approximately 2.5 min for the agarose to dissolve completely when using the microwave at 50% power. Note: Be sure to loosen the cap to the bottle before heating the solution in the microwave. Use a large volume glass bottle and observe the bottle during heating to prevent overflow of the solution as it begins to boil.
3. In fume hood, add 8.78ml formaldehyde to solution by slowly pipetting to side of bottle. Do not create bubbles.
4. While still in the fume hood, add 6.5μL of Ethidium-Bromide (final concentration 0.5μg/mL).
5. Let the agarose solution cool enough to be able to handle the glass bottle by hand.
6. Place gel tray in electrophoresis apparatus making sure that the entire tray is level and that the red gaskets on the edges of the gel tray run lengthwise along the edge of the apparatus. The red gaskets should form a complete seal around the gel tray within the casting tray.
7. Place the 20well gel comb into the slots of the gel tray.
8. Pour the cooled molten agarose (50-60°C) solution into the gel tray.
9. Allow gel to solidify at room temperature, 20-40mins. The agarose gel will be white in color when it has solidified.
10. While the gel is solidifying, make 700mL of the running buffer. Dilute 10X MOPS to 1X MOPS by mixing 70mL 10X MOPS with 630mL of RNAse free water. Add 35μL of Ethidium-Bromide (final concentration 0.5μg/mL).
11. Also while gel is solidifying, Add the blue dye (10X) to the RNA sample volume such that the final concentration of blue dye is 1X. For a 20μL sample, 2μL of blue juice is sufficient. Keep RNA sample with blue dye on ice.
12. Remove the gel tray from the casting tray and rotate it 90degrees so that the red gaskets now run in the perpendicular direction within the electrophoresis apparatus. The loading wells must be on the black anode (-) end of the apparatus. The gel should always run towards the red anode (+) end of the apparatus.
13. When the agarose gel has solidified, carefully remove the comb from the solidified gel.
14. Gently pour the 1X MOPS over the gel until it is submerged under at least 1mm of the running buffer.
15. Load your RNA sample into the wells using gel loading pipette tips. Wells can be better visualized by placing the apparatus on the black countertop for contrast.
16. For the DNA ladder, load 0.4g of the ladder per mm of lane width (3.9mm for the 20well comb, 7.2mm for the 12well comb). For 20well comb, add 15.6L of the DNA ladder (1.56g for 20well, concentration of 0.1g/L).
17. Gently place lid on apparatus.
18. Set the voltage to 100V by pressing “V” on the electrophoresis apparatus control box, press “Set”, adjust the voltage knob to 100V, and then press “Set”.
19. Turn the switch to “On” and run the gel at 100V for about 1.5hours. (Run time and voltage may vary depending on gel composition and thickness.)
20. Ensure that the blue dye front is running towards the red anode.
21. Place gel on UV transilluminator for visualization and analysis. Turn on for a couple of seconds.
22. Photograph gel with standard camera to save results.
23. Run excess MOPS buffer through an ethidium bromide extractor before disposing it down the sink. Store used extractor wrapped in plastic and log the volume of flow through.

Notes

Used in Stanford for Tissue Engineering Lab Course (ME385B)and 2007 Winter/Summer TC workshops.

Contact

• History: CRJ-CHK-SS, last updated 1/3/07

or instead, discuss this protocol.