Important information to be documented throughout this procedure

1. Number of samples run

2. What DNA ladder was used

3. What gel box was used

4. What comb type was used (how many wells)

5. Percentage of agarose concentration in gel

6. Voltage used and time duration of experiment

7. Unexpected and expected observations and results (either in writing or in picture form)

8. Clarification notes (protocol modifications, issues, significant notes)

Important notes

9. Before starting the protocol, understand how many wells and samples you will need.

10. Gel box #1 requires about 30mL of molten agarose to fit the gel tray.

11. Gel box #2 and #3 requires about 50mL of molten agarose to fit the gel tray.

12. Do not forget to add the pre-stain to the molten agarose.

13. Use the proper DNA ladder for your expected fragments. The DNA ladder reference key can be found online by searching the company and product name or number.

14. TBE may be used for the gel in substitute of TAE when a higher voltage is required. If so, be sure to use TBE as the running buffer as well.

Creating the Gel:

15. With the table above, input the desired amount of gel in mL into cell A2 (highlighted) of the table as well as the concentration of the gel stain into A4 (highlighted), without commas or punctuation.

This will provide the amounts for the 1X TAE, agarose, and gel stain needed to make the gel.

16. Measure out the 1X TAE into a graduated cylinder.

17. Using the scale, measure out agarose using a weigh boat.

Make sure the scale is tared with the weigh boat on the scale.

18. Pour half of the TAE from the graduated cylinder into a flask.

19. Add agarose into the flask and add the remaining half of the TAE.

20. Microwave flask for 30 sec and then microwave in 10 sec intervals until there is no more agarose visible. Make sure it does not boil over. The solution should be completely clear and have no powder specks. Make sure to place some paper towel in the open end of the flask so it does not bubble over.

21. Allow flask to cool, swirling with a glove every couple minutes to prevent solidification.

The gel is ready for the next step if it is, still warm, but cool and comfortable enough to hold with gloved hands.

22. Pipette the gel stain into the cooled flask. Swirl the flask around so the gel stain is well incorporated into the solution.

Pipette the gel stain directly into the solution.

23. Use the gel casting dams, or tape the ends of the gel tray to prevent the molten agarose from flowing out. Pour contents of flask into gel tray, with the gel comb in place, until it reaches the tip of the gel tray to avoid flow-over. The entirety of the solution may not be used.

24. Wait for gel to solidify (~30 minutes). Remove tape and gel comb after solidification.

Running the Analytical Gel

25. Place gel tray in gel box and fill gel box with TAE solution until gel tray is submerged, the TAE level should be close to 0.5 cm over the edge of gel tray.

Make sure that the gel box is oriented such that samples will run towards the red (positive) end of the gel box

26. Pipette 6µL of sample onto parafilm as drops, and then pipette necessary amount of loading buffer onto the samples to mix. Pipette up and down to mix.

Take care to remember which tube corresponds to which droplet.

27. Load in 6µL of the ladder into one well.

28. Load in 5µL of the sample mixed with loading buffer into each well.

29. Connect the power supply to the box, being careful to match the color of the wires.

Black knob connects with the black wire. Red knob connects with the red wire.

30. Turn on the power supply and run the gel. Run the gel at 120V for 45 minutes, or until the loading dyes are 3/4 down the gel.

Check for bubbles at each end of the gel box, which are a sign that the current is flowing.

Note: the voltage can be changed according to desired results for the experiment and should be written in notes. A lower voltage is recommended if any expected fragment will be less than 500bp. A higher voltage will be faster to run, but can cause "smiling" to occur.

Reading the Analytical Gel

The analytical gel may be preliminarily checked in the gel box with the blue light filter and blue flashlight.

31. After turning off the power supply, remove the gel tray from the gel box and slide off the gel onto the the blue light box.

Take care not to tear the gel. Work carefully.

32. Turn on the blue light box and use the lightbox filter to view results.

You may need to go into the dark bathroom to see results.

33. Properly wipe down light box and filter with a paper towel after use.

Storage + Disposal

34. The gel may be stored in a ziploc bag wrapped in moist paper towel for one week.

35. Dispose of the gel in the lab trash.