BME100 f2016:Group16 W8AM L5

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OUR TEAM

Name: Nandini Sharma
Name: Christian Forbus
Name: Jared Macanas
Name: TJ Smith
Name: Brandon Gandolf


LAB 5 WRITE-UP

PCR Reaction Report

For Part C of the lab, our group prepped our samples for the Polymerase Chain Reaction. To complete this we needed to know proper lab procedure such as how to pipette and label. The pre-lab was helpful in informing us of the steps in the process of pipetting. The first step was to label the tubes. We were able to maintain the labeling structure that we established in the initial parts of this lab. Once we had the pipette it was easy to fell the difference between the first and second stop. It takes more force to get to the second stop than it does to get to the first stop. While pipetting, it was difficult to ensure that all the liquid was all collected. It took a few tries on the first few tubes to ensure all the liquid was empties. By the end we were able to ensure that majority of the 50 microliters of the PCR mix and each sample were pipetted into the tubes. The overall process was tedious because of the amount of time it took but it was fairly simple and easy to understand.

Fluorimeter Procedure

Imaging set-up
When setting up our device to capture the images, we placed the phone, which in this case was an iPhone 7, in an upright position focused on the drop. We placed the phone approximately 7cm away from the drop and used a timer to capture the image. We used an eraser to help hold it in position while it was in the stand and then we used a folder to cover the back of the fluorimeter to keep all of the light out.


Placing Samples onto the Fluorimeter

  1. Step 1: First, we placed 80 microliters of SYBR Green 1 and 80 microliters of a calf thymus on the slide.
  2. Step 2: Then, we placed the box over the stand we created to meet up with the height of the camera.
  3. Step 3: Next, we aligned the drop to meet up with the LED light.
  4. Step 4: Then, we focused the camera on the drop.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

(1) 5 μg/mL sample

5ug-mL.png

(2) 0.5 μg/mL sample

0.5ug-mL.png

(3) zero DNA

0ug-mL.png


Calibrator Mean Values

Initial Concentration of 2X Calf Thymus DNA solution (μg/mL) Final DNA concentration in SYBR Green I solution (μg/mL) Sample Number RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation
Image 1 Image 2 Image 3
0 0 C-6 14278351 13427774 14185926 13964017 466693.702
0.25 0.125 C-5 19226506 17073922 46566069 27622165.67 16441168.13
0.5 0.25 C-4 15721858 48006477 46168607 36632314 18132286.65
1 0.5 C-3 23466266 24932157 225536611 91311678 116244512.5
2 1 C-2 19196572 17899556 21911378 19669168.67 2047239.519
5 2.5 C-1 21911378 21225181 21391583 21509380.67 357943.8481

Calibration curves

Calibrationcurve1group16.png

CalibrationCurve2group16.png


Images of Our PCR Negative and Positive Controls

(1) Negative control PCR sample

PCRnegative.png

(2) the Positive control PCR sample

PCRpositive.png


PCR Results: PCR concentrations solved

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

(Step 5 calculation)

Total Dilution Initial PCR Product Concentration (µg /mL)

(Step 6 calculation)

G16P 17325033.67 -2.112494389 12 -25.34993267
G16N 9674297.333 -3.387617111 12 -40.65140533
G161-1 11230618.75 -3.128230208 12 -37.5387625
G161-2 25825943.33 -0.695676111 12 -8.348113333
G161-3 12141144 -2.976476 12 -35.717712
G162-1 15417670 -2.430388333 12 -29.16466
G162-2 16374828.67 -2.270861889 12 -27.25034267
G162-3 10571880.33 -3.238019944 12 -38.85623933


PCR Results: Summary

  • Our positive control PCR result was -25.34993267 μg/mL
  • Our negative control PCR result was -40.65140533 μg/mL


Observed results

  • Patient 30857 : This patient appeared to be negative because the sample was clear, just like the negative control.
  • Patient 75331 : This patient appeared to be positive because the sample was fluorescent green, just like the positive control.


Conclusions

  • Patient 30857 :Patients initial concentration values were closest to positive control. So, the patients results are positive.
  • Patient 75331: Patients intial concentration values were closest to negative control. So, the patients results are negative.