AGAROSE GEL ANALYSIS OF RNA
Denaturing agarose gel electrophoresis is used to check the size and integrity of RNA preparations. RNA can form many different secondary structures which affect its mobility in an electrical field if it is not maintained in a denatured state. Formaldehyde is used to keep the RNA denatured. After electrophoresis, the RNA will be visualized by staining with ethidium bromide. Since ethidium bromide stains by intercalation into double-stranded nucleic acies, the denaturant must be removed from the gel and RNA secondary structure induced with salt after electrophoresis or the RNA will not stain.
Keep an ice bucket handy at all times and check to make sure that you have everything you need before you start. Again, you will be working with "naked" RNA so remember to wear gloves and keep your samples cold unless otherwise directed.
Safety
You will be using formamide and formaldehyde to denature your RNA during electrophoresis. Formaldehyde is a potential carcinogen so you should always handle it wearing gloves and in a fume hood. You will stain your RNA using ethidium bromide, a mutagen. Always wear gloves when handling anything containing ethidium bromide, including your gels. You will visualize your samples using ultraviolet light which is mutagenic. Always use correct shielding to protect your eyes and skin.
Equipment
Ice bucket
Racks for Eppendorf tubes
Gel apparatus (one per 4 students)
Trays for staining gels
Reagents and materials
Agarose
Sterile distilled water
10X MOPS Buffer (200 mm MOPS, 10 mM EDTA, 50 mM NaAcetate, pH 7.0 KOH)
37% (v/v) formaldehyde
1X MOPS Buffer
RNA Sample Buffer (20 mM MOPS, 1mM EDTA, 5 mM NaAcetate, 50% (v/v formamide, 2.2 M formaldehyde)
RNA sample dye
10 mg/ml ethidium bromide
Agarose gel electrophoresis
1. Remove an aliquot of 5 ug of your total RNA to a fresh, labeled 1.5 ml Eppendorf tube on ice for gel analysis and store the remaining sample at -80oC for next time.
2. On a gel plate, push the "gates" up at each end. It is very important to remember to do this otherwise the agarose will run out into the tank! Tighten the screws snugly, avoiding squashing the black O-rings.
3. Place the 6-well comb (well former) in position. Transfer the assembly to the fume hood.
4. In a 250 ml Erlenmyer flask mix:
0.5 g agarose
36 ml distilled water
5. Place a piece of Saran Wrap loosely over the top and microwave on medium-low for one minute. Remove the flask from the microwave. Caution, it will be hot. Gently swirl and check to make sure that all the agarose is dissolved. If not, microwave again for 20 second intervals and check again each time.
6. Add 5 ml 10X MOPS Buffer and swirl gently to mix.
7. In a fume hood, add 9 ml 37% formaldehyde solution, swirl to mix and immediately pour into the gel tray. Close the hood and allow to set for about 30 minutes. As the gel hardens, it becomes opaque. What percentage agarose gel have you made? ______
8. While the gel is hardening, prepare your sample for loading. Mix th following together on ice:
sterile distilled water / ? ul
RNA Sample Buffer / 10 ul
Total volume / 20 ul
9. Heat to 65oC for 5 minutes, cool on ice for 5 minutes and add 2 ul RNA Sample Dye. Vortex for 5 seconds and spin for 5 seconds to pull the sample to the bottom of the tube. Store in your ice bucket until you are ready to load the gel.
10. Squirt a little distilled water around the comb and carefully remove. Loosen the screws and lower both gates fully at both ends of the gel tray. It is very important to remember to do this or the electrical current will not be able to flow across the gel!
11. Place the gel in the electrophoresis chamber with the wells closest to the cathode (black [-ve]). The RNA has negatively charged phosphate groups (anions) and will migrate from the cathode to the anode (red [+ve]).
12. Pour about 350 ml 1X MOPS Buffer into the tank. The gel should be covered completely.
13. Load your samples. Draw you sample into a P20 tip. Load with the Pipetman upright, not at an angle, or the sample will not load evenly. Since your sample is in 22 ul, load it a half at a time. To load, stady the Pipetman with one hand, and depress the plunger slowly with the thumb of your other hand.
Sample
Volume (ul)
Plant
Owner
The first well should contain a molecular marker mixture. The other wells should be loaded with your samples. Make a note of the samples as shown above.
14. Carefully slide the lid on so that the electrodes connect. Connect the leads to the power supply. Red connects to positive, black to negative.
15. Turn on the power supply and set the voltage to 100 volts.
16. After about 10 minutes, check to see that the dye is moving in the correct direction, towards the anode (red).
17. Allow the gel to run for about 1 hour or until the bromophenol blue marker dye is about two-thirds the way down the gel.
18. Turn off the power supply. Wait one minute and remove the leads. Wearing gloves, slide the lid off the box and take out the gel plate. Be careful to keep the plate horizontal so the gel does not slide off.
19. Transfer the gel to a tray containing distilled water. Place on a shaker and shake gently for 10 minutes. Repeat twice, discarding the formaldehyde-contaminated water to the marked wast container.
20. Add a mixture of 50 ml (1X MOPS Buffer + 5 ul 10 mg/ml ethidium bromide) and stain the gel for 10 minutes on the shaker. Caution, ethidium bromide is a mutagen! Always wear gloves! All items contaminated with ethidium bromide should be discarded in the appropriate waste container.
21. Destain the gel for 30 minutes in distilled water on the shaker.
22. Carefully transfer your gel to the Fotodyne transilluminator. Caution, short wave ultraviolet light will harm your eyes! Do not observe gels unless the UV shield is in place over the transilluminator. Turn on the UV light and observe the gel. Destain the gel further if the background staining is still high.
23. Photograph your gel with a ruler next tot he left had side aligned so that the zero mark is next to the bottom of the well. The TA will demonstrate the procedure and supervise the photography. Make sure that you can see the ruler and samples the photograph. If the gel is not to be blotted, discard in the appropriate waste container. If you are going to blot the gel, save it in the destaining tray in distilled water until you are ready to proceed.
24. Note the appearance of your sample. Are there discrete bands, a smear or a blob at the bottom of the gel? What might these reflect?
25. Measure the distance migrated by each RNA in the marker mixture. On semi-log paper, plot size in kb against cm migrated from the bottom of the well (origin). Estimate the sizes of the major bands in your sample. What are the identities of these bands?