BIOLOGY 40-PROTOCOL #4
Summer, 2007
Name:_________________________________
Date:__________________________________
Protocol # 4 - Preparing a Sample of DNA and Loading and Running on an Agarose Gel
1. Use a micropipettor to pippette 1-2 ul of 6 X gel loading buffer (colorful tracking dye) into a new sterile microcentrifuge tube.
2. Add 5 ul of the solution containing the DNA sample to be run on the gel into the tube containing the gel loading buffer.
3. Briefly spin (for 2 to 3 seconds) the mixture down in the microcentrifuge to force contents to the bottom of the tube.
4. Place 10 ul of the DNA size marker in a microcentrifuge tube.* Shortly before loading, heat DNA fragments to 65 o
C for 3 minutes. This step can be omitted but will result in a reduction of the intensity of the 4361 base pair band.
5. Load 6-10 ul of the DNA size marker into the first empty lane of the gel.*
6. Load all 6 ul of the mixed sample(s) into an empty lane on the gel.
7. Close the gel box by placing the lid on top of the box. Make sure that the negative electrode (black anode) is closest to the wells containing your DNA samples. DNA is negatively charged and will migrate towards the positive electrode (red cathode).
8. Set the power source at 100 mA for 10-15 minutes only and press the “run” button.
You should see bubbles start to form in the running buffer.
9. After the 10-15 minutes, reduce the power to 65 - 75 mA and allow the gel to continue running for 1 hour.
*: The DNA marker used today contains fragments of the following sizes (in base pairs):
23130, 9416, 6557, 4361, 3000, 2322, 2027, 725, 570, and 125.
The 125 fragment is usually not visible after electrophoerisis using ethidium bromide.
The 3000 fragment typically appears more intense than other bands.