Horseshoe Crab Agarose Gel Protocol - Instructions Only

Derived from Maggie’s Gel Protocol

Gel Mold Setup

Always use small gel box and combs (can use two combs, 14 wells each, 28 wells total)

1. Use weigh boat and scoop above gel station to weigh out .75 grams of “Gel Red Agarose” powder
   *labelled Puritz on top”
2. Wash scoop in sink in DI II water
3. Measure 75 mL NEW 1x TAE buffer into Erlenmeyer flask using funnel
4. Wash funnel in sink in DI II water
5. Add gel powder to Erlenmeyer flask by folding weigh boat in half almost entirely and carefully pouring into flask
6. Swirl flask to mix
7. Put a scrunched Kimwipe in the mouth of the flask and put in the microwave for 1 minute.
8. Every 20 seconds open microwave and swirl flask. Use an orange glove because glass gets hot
9. Check flask until only a few small clear flakes are present
10. Set up gel mold by placing gel tray in gel box sideways to create a seal
11. Use level in top drawer at lab bench to make sure try is level
12. Let gel mix cool for 5 minutes before pouring into tray
13. Pour mix into mold and place comb in tray making sure thicker combs are placed downward
14. If any bubble are present, move to side of gel with pipette tip
15. Let gel harden 30 minutes

Gel Run Setup

1. Once gel is hard, take out and orient the gel with the comb side to the top of the box where the thermo scientific label is. Take the comb out of the hardened gel
2. Take ladder and dye out of -20 freezer from generuler 1kb plus DNA ladder box, the dye is called “tri track”
3. Pour enough “used” 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
4. Add 3-4μl of the appropriate ladder to the first well in the gel
5. On a piece of parafilm, make dots of 1ul of loading dye for each one of your samples.
6. 1 by 1 for each of your sample aliquoits, add 5ul of sample DNA to the dot of loading dye and pipette to mix. It will stay in a bubble on the parafilm.
7. Change pipette to 6ul to suck up the entire dot and add it to the gel
8. Make sure you have made a map in your notebook that shows the order of samples
9. Make sure gel box is set up to “run towards red”
10. Plug black cable into black insert and red cable into red insert of the gel box
11. Turn box on and make sure voltage is set to 80-100V. Set timer for 60 min

Imaging

1. Once done, turn off power source, unplug electrodes, and set up the imager. *Take the grey imager down from the shelf and remove the orange cover. Also take down the black imager shade and the small orange slide for imaging
2. Slide your gel from the tray into the center of the blue square on the imager
3. Place the black imager shade over the gel and the square and put the orange slide tile over the whole on the top
4. Turn on the light and place your phone camera on the orange square so you can take a picture of your gel through the filter
5. Once you are done with your gel, slide it off the imager and into the large black bucket in the cabinet under the gel bench. This is a non-hazardous waste container for only agarose and TAE
6. Pour the leftover TAE from the gel box into the used TAE container using the funnel kept on the gel bench
7. Rinse the gel box, comb, tray, and flask in DI type II water
8. Wipe the imager with a Kimwipe and DI type II water
9. Return all the materials you have used to where you found them