March 23

Lab 10: PCR and Gel Preparation 3/22/18

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Purpose: 

The purpose of the lab was to set up a PCR amplification procedure in order to extract DNA and prepare the agarose gel for gel electrophoresis.

Procedure:

PCR:

  1. Obtain 6 microfuge tubes that have 12.5 ul of 2X Master Mix in them and label them 1-6.
  2. Add 0.6ul of 20 uM stock COX1 primer to the first three tubes
  3. Add 0.6ul of 20 uM stock V4 primer to tubes 3-6
  4. Add 1 ul of our experimental DNA to tubes 3 and 6
  5. Add 1 ul of our control DNA to tubes 2 and 5
  6. Add 10.9 ul of water to tubes 2,3,5, and 6
  7. Add 11.9 ul of water to tubes 1 and 4
  8. Label each tube accordingly
  9. Place tubes in the rack and label the sheet to know where your group’s tubes are kept for next class

Agarose Gel preparation:

  1. Pour 90mL of DI water and 10mL of 1 x TAE into a flask
  2. Take 35mL of that mixture and put it in a new Erlenmeyer flask
  3. Add 0.6g of agarose to the flask
  4. Cover the flask with weighing paper and heat up in the microwave for 1:20 minutes on 70 power
  5. Place in a cool water bath for 5 minutes
  6. Add 2ul of ethidium bromide
  7. Assemble gel tray to prepare for the solution to be poured in it
  8. Gently pour the agarose gel mixture into the tray and be sure there are no bubbles
  9. Allow it to sit for about 30 minutes to were it is foggy in color
  10. Cover the gel with more 1 2 TAE stock to make sure it will not dry out and remove the comb once the gel is solidified

Data:

QTM data

1 Negative Control 2 Positive Control 3 Experimental DNA  4 Negative       Control 5 Positive Control 6 Experimental DNA
2X Master Mix 12.5 µL 12.5 µL 12.5 µL 2x Master Mix 12.5 µL 12.5 µL 12.5 µL
DNA 0 µL 1 µL 1 µL DNA 0 µL 1 µL 1 µL
20 µM COX1 0.6 µL 0.6 µL 0.6 µL 20 µM V4 0.6 µL 0.6 µL 0.6 µL
Water 11.9 µL 10.9 µL 10.9 µL Water 11.9 µL 10.9 µL 10.9 µL
Total Volume 25 µL 25 µL 25 µL Total Volume 25 µL 25 µL 25 µL

nanodrop data:

Nucleic Acid (ng/ul)            A260/A280        A260/A230

SAM 22 C           54.96                1.045                  0.2

SAM 22 S           138.8                  1.2                    0.407

The gel was labeled SAM section 22 and the tubes are labeled 1-6 and on the paper are labeled SAM

Conclusion:

In conclusion, the lab went well and we are hoping for better results than the last trial by making sure we followed the protocol exactly. If bad results are obtained it was not my group’s error but another error outside of what we did.


Posted March 23, 2018 by maureen_wassef1 in category Maureen Wassef

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