Running & Analyzing a Gel

Running a Gel (11/17/21)

DNA Sample Preparation to Run

1. Gather DNA samples (4 1.5mL tubes in Freezer A). The DNA ladder should be number 1. Write the volume of DNA that was used to prepare the sample and any pertinent dilutions. [Sample order & notes: 5µL Ladder, 0.1µL KCNJ6, 1µL KCNJ6, 10µL KCNJ6, KCNJ3, 6µL Ladder]. Specific notes on the Gel Analysis template.

2. Obtain Four 0.6 mL tubes (light green) and number them. Added 4 µL of MBG water (Fridge A) into each 0.6 mL tube. Then added 1 µL of DNA dye (Fridge A) into each sample tube. Finally added 1 µL of the DNA sample in its corresponding tube. The total volume of each sample should be 6 µL. [Pipettes were being weird & did not seem to be pulling up the right amount each time, so it took multiple attempts to get the "right" amount of liquid]

NOTE: The water volume should be adjusted if more or less DNA volume is used. Remember to record the specific volume of DNA used. Should be ~0.1-0.2 ug of DNA per 1 mm gel lane.

4. When the gel has set it will have a certain glimmer to it and look solid. Lift the gel tray up and out of the tank. Turn the gel around to where the rubber gaskets are not touching the tank walls. Fill the electrophoresis tank with 0.5X TBE until the liquid level is the same in both sides of the tank and the gel is covered in TBE (to create a salt bridge from negative to positive terminals).

5. Spin the samples down before loading them. Set your pipette to 6.5 µL to ensure complete transfer of the sample. Load samples according to the written order in the notebook. Do not pierce the gel. (If a trail of dye floats to the surface while loading, don’t worry. The DNA should sink into the well.) [pierced gel when loading ladder in lane 1, started ladder again in lane 4] [had pipette at 6.5 mL and mixed solution with pipette rather than spinning] [some 0.6 ml tubes had a drop of soln left]

6. Pipette 5 µL of the pre-made ladder directly into the gel lane. If the ladder is not pre-mixed, prepare a ladder sample as described in step 2. (**The DNA Ladder must be kept on ice when not in use and promptly returned to the refrigerator afterwards.)

7. Slide the tank cover on ensuring that the negative prod is on the same side as the DNA. Turn the electrophoresis machine on by flipping the switch on the front. Run the gel for 45 minutes at 160 V. [had trouble getting the machine to set & work but fingers crossed it works fine] [Lanes 4-9, 5muL ladder, 0.1 muL KCNJ6, 1 muL KCNJ6, 10muL KCNJ6, KCNJ3, 6muL ladder]

DNA Ladder: GeneRuler 1kb (Thermo Scientific)

If you load 5 ul of GeneRuler, you get the following amounts in the bands. Clear bands might require 10 ul (1 ug in total) depending on the bands for your samples. This is a ds linear DNA ladder. You can find these in Fridge A.