Agarose Gels

Protocol

For a 0.8% gel:

1. Set up gel-casting dock.
2. To a 250mL Erlenmeyer flask add 0.24 g of agarose (1 heaping green scoop) and 30 mL of 1X TAE.
3. Microwave (30sec/15sec/15sec) – want the mixture to boil twice and become clear, swirl around.
4. Allow the mixture to cool until you can place your hand on the bottom of the flask, add 3 uL of RedSafe DNA dye.
5. Pour hot mixture into gel-casting dock and place comb. Make sure no bubbles are formed. The gel will cast in about 45 mins at room temp or 15 minutes in the fridge.
6. Remove the comb.
7. When taking out the gel, untighten the knob to release the top and bottom half of the casting dock, but hold them together with your hand. Carefully slide the gel out.
8. Place the gel into the gel-running dock and cover fully with running buffer. The running buffer for a gel: 0.5X TAE => (5 mL 50X TAE + 500 mL dH₂O). Make sure the DNA is running towards the positive end!
9. Add ladder. For the small wells, add 2 μL of ladder; for the large wells, add 8 µL of ladder. The ladder can be found in the ladders box in the -20°C freezer.
10. Add the appropriate amount of 6x loading dye to the samples and then load into separate wells.
11. Run the gel at 100V for 45 minutes (35 minutes for smaller DNA <1 kB)
12. Place the gel on the UV imaging plate and place the plate into the gel imaging doc.
13. Turn the computer on and sign in.
14. Open the program "Image Lab" and press select, then "nucleic acid", then "ethidium bromide".
15. Press "run protocol", alternatively press the green button on the GelDoc once Image Lab is open.