8: Gel Loading and Running

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Nathan Reyna, Ruth Plymale, & Kristen Johnson
Ouachita Babtist University & University of New Hampshire

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Gel Loading and Running using MiniOne Instructions

Using agarose gel electrophoresis for the visualization of digested DNA.

Video example of pouring and running a gel

Reagent name	Storage Temperature
Agarose	Room Temp
1x TBE	Room Temp
SybrGreen/SybrSafe/GelRed	Room Temp (Keep in dark place)
Digested DNA	\(4^{\circ}C\)
DNA Ladder	Room Temp
Loading Dye	Room Temp

TIPS BEFORE STARTING:

Start by making a bottle of 500 ml stock of 1x TBE (C1V1=C2V2) for your team’s running buffer and to make agarose gels. (You can store this for future use- label the bottle clearly!) You will utilize either 10X, 25X or 50X TBE stock buffer– Pay attention to what stock solution you have been provided with!

Procedure:

You will make 30ml of agarose that is 1X TBE and usually 0.75-1.5% agarose. (See Appendix V for suggestions on what percentage to use.)

a. Measure ___ g of agarose (0.75-1.5%)

b. Add ___ mL of 1x TBE buffer.

Heat gel for 1+ min in microwave – (Use plastic flasks!). Swirl to make sure all crystals are in solution!
Let the gel cool for 1 minute.
Add 2-3 \(\mu L\) of SybrGreen, SybrSafe or GelRed (ask your instructor where this is stored; keep tube in the dark at all times)
Let the gel cool for another minute - Do not pour HOT!!!!!
Split warm agarose between the two gel containers (you will only need one). Do not forget the combs.
Load 5 \(\mu L\) of your DNA ladder per gel lane. (Your DNA ladder should preferably be in the first lane of your gel).
Load your samples into the wells of the gel, and mark down in your notebook the order in which you loaded the lanes. a. Loading your samples: 5 \(\mu L\) of DNA mixed with 1 \(\mu L\) of 6x loading dye. Load this directly in the well.
Run gel for 20 min in the Gel rig (set up according to diagrams below). While the gel is running, simulate the digest(s) in your lab (physical/digital) notebook, adding the Quick-Load Purple 1 kb Plus DNA Ladder as your ladder and paste the image of the expected gel in your notes. Take a picture of your actual gel results and paste them into your lab notebook.

The gel can be visualized in the gel rig (blue light) or for more sensitivity on the UV light box. WARNING: UV light is dangerous. Do not use unless you have talked to your professor!

See Appendix V for any questions you might have about the agarose gel or the electrophoresis protocol