This protocol was written by Maggie Schedl for the Putnam, Prada, and Puritz labs

1. For small gel box, insert gel tray into the box with the rubber gaskets touching the long side of the gel box. Make sure gasket forms an air tight seal with the rubber noodles. Insert desired comb size into the traygel for the gel molds.

2. Making gels:

   For a 1% gel:

   • small box: 75mL new 1X TAE buffer and .75g agarose
   • med box: 100mL new 1X TAE buffer and 1g agarose

   For a 1.5% gel:

   • small box: 75mL new 1X TAE buffer and 1.12g agarose
   • med box: 100mL new 1X TAE buffer and 1.5g agarose
3. Use a weigh boat to measure agarose and using the Erlenmeyer flask from the gel bench to measure the 1X TAE buffer
4. Add the agarose into the Erlenmeyer flask by taco-ing the weigh boat and carefully pouring the powder as to not get it stuck to the side of the flask.
5. Put in the microwave for 1 minute. Every 20 seconds open microwave and swirl flask. Use heat protective gloves because glass gets hot and don’t be afraid to check too often because it can boil over. Microwaving time can vary, so keep adding time until when you look at the liquid when swirling it is completely clear and there are no “flakes”
6. Add 5μl GelRed or 3.5μl GelGreen stain to the liquid and swirl the flask to mix thoroughly.
7. Let the gel liquid cool up to 5 minutes in the flask before pouring into the gel tray, if you can safely hold it, that’s a good sign it’s ready
8. Using a pipette tip, move any bubbles to the side of the tray
9. Let harden until opaque and cool ~30min
10. Take out and orient the gel with the comb side to the top of the box. Take the comb out of the hardened gel
11. Pour enough “used” 1X TAE buffer into the gel box to cover the gel with a thin layer of liquid and make sure there are no dimples where the wells are
12. In a new strip tube, make 9μl aliquots of your DNA sample and 11uL. Add 1μl of loading dye (Tri-Track) to each sample and vortex and spin down to mix
13. Add 3μl of the appropriate ladder (1kb GeneRuler) to the first well in the gel
14. Add your samples to each well: ~10μl. If running both DNA and RNA you can insert two combs (see figure) to run at the same time. Make sure you have made a map in your notebook that shows the order of samples
15. Make sure gel is set up to “run towards red”
16. Plug black cable into black insert and red cable into red insert of the gel box
17. Turn box on and make sure voltage is set to 80-100V. Set time for 45-60min
18. Once done, take the tray out of the boxy and using a Kimwipe, slide the gel into your hands. Bring over to the imaging plate and set in the center of the blue square
19. Place the orange shield over the gel
20. Take a cardboard box with a hole cut in it and put it over the light imager
21. Turn on the imager and using your phone take a picture of the gel through the box, turning off the lab lights helps
22. Slide the gel off when you are done and place it in the large plastic jar in the SAA for gel waste
23. Pour the leftover TAE from the gel box into the used TAE container using a funnel kept in the drawer
24. Rinse the gel box, comb, and flask in DI type II water
25. Wipe the imager with a Kimwipe and DI type II water
26. Next you want to annotate your gel image to include the sample id.

This can easily be done in something as simple as google slides

• Expectations
  • "”Good” DNA should form a distinct band a the very top of the gel.
  • “Good” RNA should form two distinct bands at the 18S and 28S locations about halfway down the lane.

  ## RNA Quality

Proceed to this step if RNA does not appear clean on the gel. If RNA quantity is sufficient follow the Zoe’s Tape Station Protocol to determine RNA quality and obtain a RNA Integrity Number (RIN). “Good” RNA should have a RIN above 8.0 and form two distinct peaks at the 18S and 28S locations.