Joyce:DNA electrophoresis

Procedure

To make a 1% gel:

1. Mass out 0.5 g of agarose
2. Place the agarose in a 250 ml erlenmeyer flask
3. Add 50 ml of 1X TBE to the erlenmeyer flask
4. Heat in the microwave for 1 minute 20 seconds
5. Let the melted agarose cool off (about 5-10 minutes)
6. While wearing gloves, add 4 ul of ethidium bromide and swirl. CAUTION: ETHIDIUM BROMIDE IS A CARCINOGEN. Always wear gloves when handling it. Always allow ample time for the agarose to cool before adding it as it will vaporize.
7. Pour into a well-taped gel case
8. Use a pipette tip to push bubbles to the side
9. Add the appropriate comb
10. Allow about 15-20 minutes to polymerize

To run the gel:

1. While wearing gloves, remove the tape
2. While still in the gel case, place the gel into the appropriate electrophoresis box
3. Slowly and gently, remove the comb
4. If necessary, add enough 1X TBE to completely cover the wells of the gel
5. Load samples
6. Place the lid on the electrophoresis box
7. Connect the box to the power source
8. Increase voltage to about 100 V
9. Verify everything is working by checking for bubbles

To stop running the gel:

1. Turn the voltage all the way down
2. Turn off the power source
3. While wearing gloves, remove the gel and place it into a container
4. Place the cover back on the electrophoresis box

Notes about the procedure

• To better resolve bands less than 1000 bp, increase the concentration of agarose up to 2%
• Try to remove as many bubbles as possible as they can prevent the migration of the DNA
• After microwaving the agarose, it's important to let it cool sufficiently (you should be able to touch it to your wrist for 10 seconds without it hurting) before adding the ethidium bromide as it would vaporize and you

Calculations

0.50 g / 50 ml = 1%

Solution

TBE