Agarose Gel Electrophoresis

Picture above: Box 13 in Freezer C; You see the 1kb DNA ladder (GeneRuler) in the yellow rectangle and the RNA ladder (RiboRuler) in the blue rectangle. DNA dyes (6x) are next to the RiboRulers.

c. RNA Sample Preparation

NOTE: Wear gloves and put Eliminase on to handle RNA. The RNA samples and ssRNA Ladder should be kept on ice when not in use.

1. Gather desired RNA samples and record the sample order and RNA volume added as explained in the DNA sample preparation protocol.

2. For each RNA sample, add 1 µL of RNA, 2 µL of MBG water (Fridge A), and 3 µL of 2X RNA Dye (Box 13, Freezer C).

3. The RNA ladder requires additional preparatory steps before it can be loaded.

a. Combine 2 μL of ssRNA Ladder (Box 13, Freezer C) with 8 μL of 2X RNA Dye.

b. Obtain a heating block and thermometer (both in Drawer B). Place the thermometer in the smallest hole and turn the ON/OFF switch to high.

c. Turn the high temperature control dial up to increase the temperature. Adjust the temperature according to the following settings: Incubate at 90°C for 2 minutes.

d. Immediately place it on ice for 2 minutes and load the entire volume into its corresponding well.

4. When the gel has set it will have a certain glimmer to it and look solid. Lift the gel tray up and out of the tank. Turn the gel around to where the rubber gaskets are not touching the tank walls. Fill the electrophoresis tank with 0.5X TBE until the liquid level is the same in both sides of the tank and the gel is covered in TBE.

5. Spin the samples down before loading them. Set your pipette to ~6.5 µL to ensure complete transfer of the samples. Load samples according to the written order in the notebook. For the RNA ladder, load the entire volume into the well. Do not pierce the gel.

NOTE: If a trail of dye floats to the surface while loading, don’t worry. The RNA is dense and should sink into the well.

6. Slide the tank cover on ensuring that the negative prod is on the same side as the DNA. Turn the electrophoresis machine on by flipping the switch on the front. Run the gel for 90 minutes at 60 V.

RNA Ladder: RiboRuler (SM1823, Thermo Scientific)

If you load 4 ul of RiboRuler you get the following amounts in the bands. You may need to load 8 ul or more to see clear bands.

d. Analyzing Gel Electrophoresis Results

1. Remove the UV tray from the Gel Doc EZ Imager and inspect for cleanliness. If the tray is not completely clean, it can be lightly sprayed with distilled water and wiped down with a Kimwipe.

2. Don a pair of gloves and remove the gel from the tray and drain excess buffer back into the tank. Place the gel on the UV tray straight with the wells at the top and the ladder on the right. If air bubbles form, you may press them out toward the edges of the gel. Press the gel gently as too much force can break it. When done, rinse your gloves with water and dry them.

3. Place the tray into the imager and flip the power switch on, which is located on the bottom right side toward the front.

4. Open the Image Lab program on the desktop. On the window that pops up, under Protocols, click New. Under Application select Nucleic Acid Gels and then Ethidium Bromide. Click the green Run Protocol button.

5. Observe the gel image. If the bands are too faint or too strong, you may need go back and adjust the exposure time until an acceptable image is produced. To do that, go back to the Gel Imaging Window. In the dropdown box under Image Exposure, you can select Faint or Intense Bands to optimize imaging for your bands. Click Run Protocol again.

6. Once you have taken a good image, direct your attention to the Analysis Tool Box on the left of the screen. Click on the first button, Image Tools. Click Crop and adjust the cropping area to include the gel only. Once selected, right-click the gel and click Crop.

7. In order to improve the contrast, select the Image Transform button above the picture of the gel. It looks like a half white and half black sun. Under Options toward the bottom, click Invert Image Display. The black and white areas of the gel will now be inverted.

NOTE: If you are running only PCR or RNA products, you will not need to create any bands for the experimental or reference bands. Skip ahead to Step 17.

8. Click the Lanes and Bands button under the Analysis Toolbox on the left of the screen. Under Lane Finder click Manual. You will be prompted to enter the number of lanes desired. Enter the number of lanes that you loaded including the DNA ladder and click OK.

9. A box should appear with the lanes. Use the tools on the left to move and resize the lanes, so that they are centered on the bands/smears. Note that there are tools to adjust all or a single lane. The tools are under All Lanes and Single Lane, respectively.

10. Once the lanes have been properly adjusted, click the Bands tab, which is near the top of the left side bar. Under Bands click Add. Click any band on the gel to tell the program which bands will be analyzed. A single sample may have a more than one band, and they should all be selected. Note there is a Delete button in case you make a mistake. Simply click the band you want to delete to remove it.

NOTE: PCR product samples and RNA samples do not need bands created for them.

11. When you add a band, you will notice that three lines are created: one solid line that runs through the band of interest and two dashed lines, one above and one below the solid line. The dashed lines mark the upper and lower boundaries of the band. In case you have a clearly defined band, the dashed lines will automatically be created on the top and bottom edges of the band. In the case of a smear, most likely the dashed lines will need to be adjusted to correctly mark the edges of the smear. You can do so by clicking Adjust under Bands on the left side bar. To adjust the dashed lines simply click, hold, and drag the line to its new location.

12. Click the green arrow box at the top right of the sidebar to return to the Analysis Tool Box. Click the button named Quantity Tools. Switch from the Relative tab to Absolute tab at the top. Find the NEB 1 kb DNA Ladder Reference in Drawer A. It can also be found online.

13. Back in Image Lab, click Select under the Absolute tab. Click the first distinguishable band in the ladder lane. A box will come up prompting you to enter the mass value of the band. Enter the mass amount from the card corresponding to the band you have selected and click OK. Repeat for the rest of the bands.

NOTE: Some reference bands may overlap each other, making it difficult to identify and select them individually. Try your best to select them individually and assign the correct corresponding mass values.

14. Once all reference bands have been labeled, the computer can start estimating the DNA masses in the bands. Click the unknown bands. A box will come up showing the estimated mass of DNA present. Write this value with correct units, rounded to the nearest ones place, in the lab notebook next to the sample’s name. To analyze the next band, either click Cancel or close the box, but DO NOT click OK, as this will make the unknown band a reference band.

15. If more than one band is present for the same sample, write the first amount followed by a plus sign. Click the next band and record the new amount. Repeat until all bands have been recorded in the lab notebook and write an equal sign at the end. Add all the individual values and record the result.

16. Once all lanes have been analyzed. Divide the DNA mass values or the sum thereof for multi band lanes by the corresponding volume of DNA solution loaded on the gel. The result is the concentration of DNA. Use appropriate units.

17. Click the green arrow box once again to return to the Analysis Tool Box. Click the Annotation Tools button and under Add Annotations click Text. Add annotations by clicking anywhere on the gel. Label the lanes with the name of the DNA/RNA listed in the lab notebook, concentration (if applicable), and whether the reaction succeeded (if applicable). See the table below for examples. Rotate labels by clicking the 90° to the Left, under Rotate.