2024 Lab 1-Pipetting-Electrophoresis Gel-
Benchling
Purpose:
The purpose of this lab is to familiarize ourselves with pipetting, analyzing and conducting an electrophoresis gel to gain
experience for future labs.
Experiment 3: Electrophoresis gel
Protocol
Materials
• 1 Erlenmeyer flask (150mL)
• Graduated cylinder (100mL)
• Electrophoresis gel box (see set up on the diagram)
• 1X TBE buffer
• Agarose
• SybrSafe (nucleic acid staining solution)
• Ladder (1 Kb Plus DNA Ladder, Invotrogen)
• DNA of unknown size
• Loading Dye 10X
• H2O
Methods
1. Weigh enough agarose for a 25mL gel at 1% concentration and add it to an Erlenmeyer flask
2. Using the graduated cylinder measure 25mL of 1X TBE buffer, add it to the Erlenmeyer
flask and shake gently
3. Microwave the solution for ~25sec or until it bubbles. If you see bubbles forming, remove
the flask from the microwave and shake it gently to dissolve the agarose. Microwave a
few extra seconds until it bubbles and you don't see any agarose crystals in the flask. Be
careful not to burn your hands
4. Let the flask cool down a little, add 2.5μL of SybrSafe and shake gently
5. Set up the gel box, casting damns and place the 8-wells comb (near the negative charge
side) and pour the agarose in the gel box
6. Let the agarose solidify for 30min
7. Remove the casting damns, the 8-wells comb and pour the 1X TBE buffer into the box
until just covers the top of the gel
8. On a parafilm, prepare 12μL of sample as follow:
i. 8.8μL H2O
ii. 1.2μL Loading Dye 10X (pipet-mix)
iii. 2μL DNA (pipet-mix)
9. You can also prepare up to six 12μL blank samples
by omitting the DNA: This is just to practice loading.
10. Load the gel by first inoculating 6μL of the 1Kb
ladder directly into well 1
11. Load your 12μL DNA sample into well 2 and all your blank samples into wells 3-8
12. Plug the gel box to the power source (black chord connected to anode and red chord
connected to the cathode)
13. Run the gel for 1h at 100V to migrate the DNA.
Gel observation
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