Transforming Styrofoam waste into biodegradable plastic

Safety Considerations: Ethidium Bromide (EtBr) is carcinogenic. Avoid skin contact and do not inhale. Prepare gel within hood specifically used for EtBr work, and dispose of all waste which contacted EtBr within EtBr labeled water container (gloves, pipette tips, gel tape, etc).

Procedure:

1. Tape the open edges of the gel mold to prevent leakage. Double Tape firmly.

2. For a 1% gel, add 0.4g of agarose to 50 mL of 1X TAE. Swirl flask. Microwave in 30 second intervals swirling in between until agarose is completely dissolved. Limit the amount of bubbling as much as possible

1. If you have a 50X stock solution of TAE, create 350 mL of 1X TAE at a time. 50 mL will be used for gel and 300 mL will used as a running buffer.

2. Use a 1.2% to 2% gel for larger DNA pieces

3. Let mixture cool until it is warm to the touch of your skin

4. Under the hood, add 1.0 uL of EtBr to the flask and swirl until dissolved

5. Pour mixture into gel mold and place comb into the mold

6. Let the gel cool until it is solid

7. Gel can be stored at 4C for ~10-15 days if it needs to be used later

Running the gel:

Procedure:

1. Remove tape from gel mold and place in gel box.

2. Pour 300 mL of 1X TAE into gel box

1. If using a seaplaque gel, BE EXTRA CAREFUL. Do not pour TAE directly onto gel; pour it into the wells in the sides

3. Gently remove the comb from the gel

4. On a piece of parafilm, aliquot correct amounts of dye onto the parafilm for each thing you want run on the gel. Final concentration should be 1X of the dye. (i.e. for a 6X loading dye, you would add 1 uL of dye and 5 uL of DNA)

5. Add the correct amount of DNA to the aliquoted dye. Mix thoroughly by pipetting up and down on the parafilm. To limit bubbles, pipet gently.

6. In one well, add 10 uL of DNA Ladder

1. Gently place pipet tip in well and slowly pipet in, limiting the amount of bubbles as much as possible

7. Add the DNA+Dye products to the other wells. Use a piece of tape to label where everything is.

8. DNA is negative so it will run to the positive side. Black is negative and red is positive. Place cap on the gel box so that the DNA will flow from negative to positive

9. Run the gel at 120 volts (for seaplaque gels, run at 90 volts)

1. more bubbles should rise on the side of the gel wells when first started than the far side

10. Stop running the gel after it travels three-fourths of the way

1. Sometimes you might need to run longer or shorter depending on the DNA lengths

11. When removing gel to check under UV light, gently pick up gel mold and pour out remaining TAE

1. Be very  careful with seaplaque or other preparatory agarose gels since they break very easily