BME100 f2017:Group1 W1030 L5

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Scaredy Pants

Emily Hanzlick
Abby Krell
Marissa Morales
Anthony Silva
Matias Fernandez


LAB 5 WRITE-UP

PCR Reaction Report

In a Polymerase Chain Reaction (PCR), a specific sequence of DNA is amplified and multiplied. PCR requires Taq DNA Polymerase, deoxyribonucleotides, template DNA, primers, and a reaction buffer. In this lab we analyze PCR reactions to test two patients for the presence of a known pathogen. The process begins with the mixing of the template DNA, Taq Polymerase, and primers into a tube. The tubes are then sent to a thermocycler to be heated to separate the DNA, from there the primers for the target DNA bind and amplify the segments of the targeted DNA. This makes it possible to detect the presence of the pathogens in the patient DNA samples.

During the setup of our reaction, the pre-lab was very informative and helpful in explaining how to complete the micropipetting for both patients and their replicate DNA. We had trouble differentiating between the first and second stop, though it only took a few tries to get the hang of it, and then it was easy to complete the rest of the setup. We were careful in labeling so that we did not have to go back to our first write-up and change the labeling scheme. There were no errors in labeling, and the setup went well overall. The final reactions had the same amount of liquid, and there was a very small amount of liquid left in the tubes that held the DNA samples and PCR reaction mix.

Fluorimeter Procedure

Imaging set-up

The following procedures were completed during the PCR experiment. First, a fluorimeter kit was obtained, including: black box, fluorimeter, phone stand, glass slides. Then, two green micro test tube containers were placed on top of each other in order to get the fluorimeter to line up with the phone camera. The glass slide was placed inside the fluorimeter from the back with the glass slide facing down and the rough. hydrophobic side with the dots going towards the front of the fluorimeter. The camera used was an iPhone 6, with the flash settings turned off. The iPhone was held up using the phone stand that came with the fluorimeter kit. The black box was used to create a dark environment for the iPhone imaging.


Placing Samples onto the Fluorimeter

  1. Set pipette to 80 microliters
  2. Pipette 80 microliters of the desired DNA sample (water, 0.25, 0.5, 1.0, 2.0, 5.0) and then drop it onto the middle of the glass slide
  3. Remove pipette tip and get a new tip
  4. Pipette 80 microliters of SYBR Green 1 solution and then add it to the drop on the middle of the glass slide
  5. Move the black box over the fluorimeter set-up to create a dark environment, then use the iPhone to take three images of the drop, with the drop in focus.
  6. After three images of the drop are taken, all the liquid is pipetted off and then disposed of in a waste beaker.



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

ImageJ: Zero DNA

Calibration0.PNG

ImageJ: 0.5 μg/mL sample (low calf thymus DNA)

Calibration0.5.PNG

ImageJ: 5.0 μg/mL sample (high calf thymus DNA)

Calibration5a.PNG

Calibrator Mean Values

Table 1: Calibrator Raw Data

Sample Number Image Number Final DNA concentration in SYBR Green 1 solution (µg/mL) Area Mean Pixel Value RAWINTDEN of the drop RAWINTDEN of the background
Calibration 0.0 1 2.5 47956 86.441 4145365 200938
2 2.5 47956 82.417 3952380 202200
3 2.5 47956 82.699 3965899 213770
Calibration 0.25 1 1 47956 113.946 5464414 209035
2 1 47956 113.569 5446291 208512
3 1 47956 115.439 5536011 204692
Calibration 0.5 1 0.5 47956 149.951 7191061 213014
2 0.5 47956 149.709 7179447 208211
3 0.5 47956 152.512 7313874 213955
Calibration 1.0 1 0.25 47956 214.836 10302664 208531
2 0.25 47956 210.562 10097702 201800
3 0.25 47956 216.349 10375213 201307
Calibration 2.0 1 0.125 47956 238.391 11432268 208362
2 0.125 47956 234.668 11253742 229573
3 0.125 47956 234.405 11241115 226871
Calibration 5.0 1 0 47956 238.327 11429210 213803
2 0 47956 238.483 11436712 212428
3 0 47956 237.795 11403679 210607


Calibration curves

Dotplot1final.PNG

Dotplot2final.PNG


Images of Our PCR Negative and Positive Controls

ImageJ: Positive Control

Positivecontrola.PNG

ImageJ: Negative Control

Negativecontrola.PNG

PCR Results: PCR concentrations solved

Table 5: PCR Solved

PCR Product Tube Label Mean of RAWINTDEN Drop - Background PCR Product Cocentration (µg/mL) Total Dillution Initial PCR Product Concentration (µg/mL)
Positive Control 10980608 72240833.16 12 866889997.9
Negative Control 4281269.667 28166238.86 12 337994866.3
Patient 1 - Sample 1 4395681.333 28918947.19 12 347027366.3
Patient 1 - Sample 2 6176889.333 40637420.88 12 487649050.5
Patient 1 - Sample 3 2594612.333 17069809.04 12 204837708.4
Patient 2 - Sample 1 11699727 76971879.21 12 923662550.5
Patient 2 - Sample 2 10974659.67 72201699.39 12 866420392.6
Patient 2 - Sample 3 11208122.67 73737640.18 12 884851682.1


PCR Results: Summary

  • Our positive control PCR result was 866889997.9 μg/mL
  • Our negative control PCR result was 337994866.3 μg/mL


Observed results

  • Patient 54647: These samples when qualitatively observed, look similar to the negative control sample results because there was very little green in the drops when observed in the dark. When quantitatively observed, the sample results for this patient are also very similar to the negative control sample because the initial PCR Product Concentration for the negative control is 337994866.3 μg/mL and the average initial PCR Product Concentration for this patient is 346504708.4 μg/mL which are very similar values.
  • Patient 67052: These samples when qualitatively observed, look similar to the positive control sample results because there was a lot of green in the drops when observed in the dark. When quantitatively observed, the sample results for this patient are also very similar to the positive control sample.When quantitatively observed, the sample results for this patient are also very similar to the positive control sample because the initial PCR Product Concentration for the positive control is 866889997.9 μg/mL and the average initial PCR Product Concentration for this patient is 891644875.067 μg/mL which are very similar values.


Conclusions

  • Patient 54647: Negative; This patient does not have the disease because their samples show little to no evidence of having the DNA sequence associated with this disease.
  • Patient 67052: Positive; This patient does have the disease because their samples show evidence of having the DNA sequence associated with this disease.