BME100 f2017:Group11 W1030 L5

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OUR TEAM

Name: Teleah Hancer
Name: Jose vallejo
Name: Una
Name: Zoe Marmitt
Name: student
Name: student


LAB 5 WRITE-UP

PCR Reaction Report

Going through the pre lab material was not only extremely important to understand how to pipette, but it made the lab run smoothly. Doing the PCR virtual lab helped explain the reason why we are pipetting. The virtual micro-pipetting exercise familiarized us with the micro pipettor.We all practiced using the pipette with no liquid to understand the difference between the first and second stop. Practicing micro-pipetting allowed us to be more precise when mixing the PCR primer mixture and the DNA, and ensured that all the final reactions had the same amount of liquid. To double check that the amounts were the same, we checked to make sure there was no liquid left in the tubes that the DNA samples and PCR reaction mix was taken from. Since everything was properly labeled at the start of the lab, we did not have to change our labeling scheme.

Fluorimeter Procedure

Imaging set-up

It was important to put on all PPE before starting. Then all the materials were gathered. This included the fluorimeter, 3 glass slides, an iPhone 6, 3 small pipette holder boxes. First, one side of the fluorimeter box was opened and the lid was removed so everything could be easily set up. Then the smooth side of the slide was put face down in the fluorimeter. Next, the phone camera was turned on and set to a 3 second timer. It was then placed on the phone holder and an 80 microliters sample of H2O was pipetted on the first 2 clear circles in the middle of the slide. Then 3 small pipette holder boxes were placed beneath the fluorimeter to get the droplet level with the camera, and the camera distance was adjusted so a clear image of the droplet side could be taken. Once the camera was clearly focused on the H2O sample, the H2O was removed and the fluorimeter was ready for imaging. The lid of the fluorimeter was put on every time a picture was taken to prevent any source of light getting in.

Placing Samples onto the Fluorimeter

  1. Set the micro pipette to 80 micro liters
  2. Put on a clean pipette tip
  3. Push plunger button to first stop
  4. Insert tip into SYBR Green I solution
  5. Slowly release plunger button until all 80 micro liters are inside the pipette
  6. Place all 80 micro liters of SYBR Green I solution on first 2 clear circles in the middle of the slide by pushing plunger button all the way down to the second stopper
  7. Eject pipette tip into discard cup
  8. Put on new clean pipette tip
  9. Push plunger button to first stop
  10. Insert into sample/calibration solution
  11. Slowly release plunger button until all 80 micro liters are inside the pipette
  12. Place all 80 micro liters of sample/calibration solution over first drop
  13. After the pictures have been taken, suck up the whole drop on the slide using pipette
  14. Eject pipette tip into discard cup
  15. Adjust slide to the center of the next two holes
  16. Change slide after all five possible measurement positions have been used
  17. Repeat steps 2-16 until all calibration solutions have been put on the fluorimeter
  18. Repeat steps 2-16 until all DNA sample solutions have been put on the fluorimeter



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL sample
0.5 μg/mL sample
Zero DNA


Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND Image 1 RAWINTDEN DROP - BACKGROUND Image 2 RAWINTDEN DROP - BACKGROUND Image 3 MEAN Standard Deviation
5 2.5 C-1 17926243 16631330 16348318 16968630 841302.8364
2 1 C-2 14445901 13255933 13720561 13807465 599725.0947
1 0.5 C-3 12373343 9617279 10469292 10819971 1411100.347
0.5 0.25 C-4 10345440 12265350 12057955 11556248 1053705.74
0.25 0.125 C-5 7173099 6753052 7109988 7012046 226504.5212
0 0 C-6 0 0 0 0 0



Calibration curves

Calf Thymus DNA Calibration Curve 1
Calf Thymus DNA Calibration Curve 2

Images of Our PCR Negative and Positive Controls


PCR Results: PCR concentrations solved

Negative Control

Negative.PNG

Positive Control

Positive.PNG

PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP-BACKGROUND) PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration

(µg /mL)

G11 1-1 6991641.667 0.49582 12 4.77994
G11 1-2 28544989 11.272495 12 135.2699
G11 1-3 7234025 0.6170125 12 7.404
G11 2-1 4515353 -0.7423235 12 -8.90788
G11 2-2 7005001 o.5025005 12 6.030006
G11 2-3 7731292.33 0.8656165 12 10.3877
G11 + 16592518.33 5.296259165 12 63.555
G11 - 5221622 -0.389189 12 -4.670



PCR Results: Summary

  • Our positive control PCR result was 63.555 μg/mL
  • Our negative control PCR result was -4.670 μg/mL


Observed results

  • Patient 43374: The patients results of the PCR Product were all positive and well above O. G11 1-2 however, seems to have way more concentration than the others so that may have been caused because an error in the lab. The pictures of their droplets all seem to relatively clear with little to no white shading in it.
  • Patient 37706:The droplets seem to have a darker shade especially in the lower region of the droplet. The data for this patient only had one concentration that was negative and the rest was positive. All the PCR product concentration are close to zero except for

Conclusions

  • Patient 43374 :They tested positive for the disease because the result are very similar to that of the positive. the mean is close to the positive.
  • Patient 37706 :They tested negative for the disease because they had results similar to the negative results