BME100 f2016:Group3 W1030AM L5

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OUR TEAM

Name: Ravyn Ogden
Name: Madeleine Howell
Name: Nya Griffin Ulibarri
Name: Kaycee Bicshoff
Name: Lauren Hudson

LAB 5 WRITE-UP

PCR Reaction Report

The pre-lab reading and activities proved to be very helpful. The majority of the members of our group had little to no experience using a micropipette prior to this lab. However, after reviewing the pre-lab materials everyone felt comfortable using a micropipette. The difference between the first and second stop on the pipettor was easy to understand. In order to suck the liquid up into the pipettor, the button was pushed to the first stop. The second stop was pushed to release the contents of the micropipette into a second vessel. To the naked eye, the final reactions all appeared to have the same amount of liquid. (All the PCR tubes appeared to have the same amount of liquid in them.) The experimenters made sure that the pipettor was calibrated to the correct volume as well as making sure that all the liquid from the pipettor was released into the container. All the contents from the and PCR reaction mix were used. The labeling scheme did not change. The labeling scheme did not change. Each empty tube was labeled with the patient ID or positive/negative control and a number (1,2 or 3) to specify the replicates of the patient ID reactions.

Fluorimeter Procedure

Imaging set-up

A smart phone was placed in a cradle. The back of the phone was 6 centimeters away from the drop itself. The fluorometer was placed on two plastic trays so that the camera was level with the drop. This way, the camera was able to take pictures of the drop from the side. The camera’s flash was turned off. The whole apparatus was placed in a dark box.

Placing Samples onto the Fluorimeter


  1. The slide was placed into the fluorimeter with the smooth side of the slide face down.
  2. 80 microliters of SYBR green was placed in between the first and second circles on the slide. 9.The fluorimeter light was turned on. The light went through the drop, if the light did not go through the drop, subsequent adjustments were made.
  3. 80 microliters of calibration solution were placed on top of the 80 microliters of SYBR green already on the side.
  4. The fluorometer was then covered by the lightbox.
  5. The images taken with the camera timer were blurry. In order to increase the quality of the pictures, the fluorometer and smartphone were pushed the end farthest from the opening of the lightbox. By doing this, the fluorometer was in the darkest place in the box.
  6. A group member would position the smartphone in the cradle 6 centimeters away from drop and took 3 pictures of the drop.
  7. After the picture was taken, the drop was removed from the slide.
  8. The slide was then moved to the next position: the second and third row of circles.
  9. This process was repeated. However, for each trial 80 microliters were taken from one of the tubes labeled either negative, positive, patient ID: 41134 (1,2, and 3) or patient ID: 45645 (1,2, and 3.). The 80 microliters from one of these labeled tubes from the previous lab was placed on the 80 microliter SYBR green drop on the slide. Three individual pictures were taken of each drop.



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

zero DNA
0.5 μg/mL sample
5 μg/mL sample


Calibrator Mean Values

Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND ' ' MEAN Standard Deviation
Image 1 Image 2 Image 3
2.5 C-1 11375831 10689340 12255210 11440127 784912.5403
1 C-2 5984299 6963008 8097946 7015084.333 1057785.36
0.5 C-3 9101386 53972676 9119963 24064675 25901090.31
0.25 C-4 6703318 6194381 8617898 7171865.667 1277893.325
0.125 C-5 6349909 6388464 7092025 6610132.667 417776.0018
0 C-6 4403723 4403723 4403723 4403723 0


Calibration curves

1030 G3 CalibrationCurve1.png
1030 G3 CalibrationCurve2.png

Images of Our PCR Negative and Positive Controls

Positive Control
Negative Control

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)
positive 9386470.33 0.38647033 12 4.63764396
negative 3254425.33 -5.74557467 12 -68.94689604
41134 (1) 9846700.33 0.84670033 12 10.16040396
41134 (2) 10250326.7 1.2503267 12 15.0039204
41134 (3) 11514812.7 2.5148127 12 30.1777524
45645 (1) 3523294.67 -5.47670533 12 -65.72046396
45645 (2) 3698056.33 -5.30194367 12 -63.62332404
45645 (3) 2462140.67 -6.53785933 12 -78.45431196


PCR Results: Summary

  • Our positive control PCR result was 4.63764 μg/mL
  • Our negative control PCR result was -68.94689 μg/mL


Observed results

  • Patient 41134: Images had a bright green, fluorescent glow and an average initial PCR product concentration of 18.44736 μg/mL
  • Patient 45645: Images did not have the green glow and had an average initial PCR product concentration of -69.26603 μg/mL


Conclusions

  • Patient 41134 : concluded to be positive because the average initial PCR product concentration of the patient is 18.44736 μg/mL which is closer to the positive control value of 4.63764 μg/mL than the negative control value of -68.94689 μg/mL. Because the patient’s results are close to that of the positive control, the patient is concluded to be positive for the presence of the DNA.
  • Patient 45645 : concluded to be negative because the average initial PCR product concentration of the patient was -69.26603 μg/mL which is closer to the negative control value of -68.94689 μg/mL than the positive control value of 4.63764 μg/mL. Because the patient’s results were close to that of the negative control, the patient is concluded to be negative for the presence of the DNA.