Agarose Gel 3/22/18
Date/title: 3/22/18 Making Agarose Gel (part 2)
Objective: Set up the agarose gel so we can perform the electrophoresis and read the DNA.
Materials:
- 1xTAE
- Erlenmeyer flask
- Ethidium Bromide
- PCR product
- Loading Buffer
- Agarose
- DI water
Procedure:
- Add 35 mL 1xTAE to 0.6g Agarose in small Erlenmeyer flask. (add 90 mL of DI water)
- Cover lightly with weighing paper and a loose-fitting cap.
- Heat until solution is clear and small bubbles come off the bottom when gently swirled.
- Allow to cool until the flask is comfortable to hold.
- Have your TA add 2 µL ethidium bromide, swirl gently.
- Set up gel electrophoresis box, making sure the open ends are sealed.
- Pour agarose gel smoothly into prepared mold, with as few bubbles as possible. Allow to solidify for at least 25-30 minutes.
- Cover gel with prepared 1xTAE buffer solution so that it will not dry out.
- Carefully remove the comb and turn gel so that the wells are furthest away from positive electrode.
- 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.
- 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.
- Image with UV light.
Data:
| Tubes | Negative control (1) | Positive control (2) | eDNA (3) |
| DNA template (µl and total ng) | 0 (mL) | 1 µl | 1 µl |
| 2X master mix (µL) | 12.5 | 12.5 | 12.5 |
| 10 µM COX1 Primers | 0.63 µl | 0.63 µl | 0.63 µl |
| Water (µL) | 11.87 µl | 10.87 µl | 10.87 µl |
| Tubes | Negative control (3) | Positive control (4) | eDNA (5) |
| DNA template (µl and total ng) | 0 (mL) | 1 µl | 1 µl |
| 2X master mix (µL) | 12.5 | 12.5 | 12.5 |
| 10 µM V4 Primers | 0.63 µl | 0.63 µl | 0.63 µl |
| Water (µL) | 11.87 µl | 10.87 µl | 10.87 µl |
Mistakes:
A mistake could be not calculating the correct amount of agarose in grams that must be added to make the gel. Another mistake would be not ensuring that the comb does NOT touch the bottom of the gel electrophoresis box.
Results:
Our eDNA was 1.098 ng and our control was 1.03 ng. The goal is to obtain a 260/280 ratio and we want a ratio that’s on the higher side.
Conclusion:
Obtaining the 260/280 ratio is important because it means that we will have pure DNA which is what we want. The next step would be to perform the gel electrophoresis and see if we can isolate and identify the DNA.
Storage/label: Stored on the blue rack in the lab room. Labeled CKL and they also have the corresponding numbers on them that align with the data table (see data portion of blog).