Learning Objectives
At the end of the activity, you will be able to:
Abilities
Run a gel electrophoresis and interpret the results.
Skills
Using Benchling's software to run a virtual digest of plasmids
Running a gel electrophoresis
Instructions
To prepare an 10-lane 25ml 1% agarose gel:
Set up you the electrophoresis chamber.
Mix 25 mL of 1X TAE buffer with 0.25 g agarose in an Erlenmeyer flask and microwave the mixture for 20 seconds, stir, then microwave again for 10 seconds, stir, and microwave for a final 10 seconds. Use a heat glove when swirling the flask
Remove the Erlenmeyer flask from the microwave using a heat glove. Let the flask cool in the air for a few minutes
Cool the outside of the Erlenmeyer flask under running water while lightly swirling the liquid agarose mixture in a continuous circular motion. Make sure not to get any water inside the flask, as this will lead to impurities in the gel/improper formation. To test if the flask is cool enough to pour, touch it to the back of your wrist and if its at a temperature that you think you could feed it to a baby then you are good.
Add 2ul Sybr Safe to flask and continue to swirl the mixture for another min.
Pour the agarose mixture into the mould.
Remove bubbles using pipette tip, add comb into comb slot and allow gel to harden (usually 15 to 20 min).
To set up the gel electrophoresis:
Once the gel has hardened, remove the black mould seals and add 1X TAE solution to cover the gel. You want to add enough TAE solution so that the level of liquid is level throughout the chamber and that there is only a thin layer of solution over the gel (should be around 125mL in our case).
Remove the comb from the gel
Retrieve the PCR tubes containing the digest reactions from the incubator.
If necessary, spin the tubes for 5 seconds in a table top mini centrifuge to collect the liquid at the bottom of the tube.
Retrieve a concentrated loading dye solution (in this case, purple DNA loading dye which can be found in the electrobox beside the Sybr Safe).
In a tube rack, open all the tubes and use a pipette to place 4μl loading dye inside the tube cap. Normally you want to add loading dye in a 1:5 ratio with the sample you want to run (ie if you have a 20uL sample you would use 4uL of dye)
Close the tubes and spin for 5 seconds in a table top mini centrifuge to collect the liquid at the bottom of the tube.
Use a pipette to carefully transfer 8μl from each tube into its corresponding well in the gel that was created by the comb. Do not touch the bottom of the well, and make sure that the sample doesn't bleed into the adjacent wells. (Hold the pipette at a 45* angle, and use your non dominant hand to stabilize the pipette tip). This step takes practice to get perfect. Make sure the tip is in the well but that it is not stabbing the agar or pressed against the walls of the well.
Add 4uL of the DNA Ladder to the first well in the gel
Place lid on top to connect the circuitry of the machine. Make sure the red and black wires and knobs match.
Set gel electrophoresis machine to 100V for 45min. MAKE SURE TO PRESS “RUN”.
After gel is finished running, take a picture of it in the common tech room
Watch this short video before reading through the exercise to get an overview on how to run a digest.
