PCR/Gel Electrophoresis 2/22/2018

Materials:

• 1xTAE
• Erlenmeyer flask
• Ethidium Bromide
• PCR product
• Loading Buffer

Procedure:

1. Add 40 mL 1xTAE to 0.6g Agarose in small Erlenmeyer flask.
2. Cover lightly with weighing paper and a loose-fitting cap.
3. Heat until solution is clear and small bubbles come off the bottom when gently swirled.
4. Allow to cool until the flask is comfortable to hold.
5. Have your TA add 2 µL ethidium bromide, swirl gently.
6. Set up gel electrophoresis box, making sure the open ends are sealed.
7. Pour agarose gel smoothly into prepared mold, with as few bubbles as possible. Allow to solidify for at least 25-30 minutes.
8. Cover gel with prepared 1xTAE buffer solution so that it will not dry out.
9. Carefully remove the comb and turn gel so that the wells are furthest away from positive electrode.
10. Using a micropipette, add 5 µl of the ladder and 10 µl of each PCR product + loading buffer. If the loading buffer is not included in the Taq polymerase used in the PCR, add 5µl loading buffer to the PCR product and mix thoroughly before transferring to the gel.
11. After you have loaded your sample, place the lid on your box and turn on the power supply to approximately 100 volts. Allow to run for 30 minutes or more, allowing the loading dye to run approximately halfway across the gel before turning off the power.
12. Image with UV light.

Data:

What did not work (Ludox and cell washing): There was not very much DNA found in any of our samples after the previous steps in this experiment.

There is no data to report since we were not able to finish this portion of the experiment.

Conclusion:

The next step would be to put our DNA into the cell wells containing the gel electrophoresis. We will also need to image with UV light. This will enable us to start the process of reading the DNA.

Storage:

Our sample is on the rack and it is labelled CKLMKC