BME100 f2017:Group4 W0800 L5

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OUR TEAM

Name: Ally Coen
Name: Jose Loya
Name: Monica De La Cruz
Name: Christian Cuciniello


LAB 5 WRITE-UP

PCR Reaction Report

The pre-lab helped the group understand the process of pipetting the samples to set up the reaction. It's a simple process to run a fluorimeter analysis but also requires the small details to be in place such as making sure you have the camera about 4cm away from the drop, placing a 80uL drop of SYBR green I solution on the smooth side of the slide, placing 80uL of your sample on top of the drop of SYBR green I, and making sure to place the light directly in the middle of the drop. It was also useful learning to use the first and second stop of the pipet. The first stop allows for the exact liquid to be collected by the pipet from the sample that you selected, but if you go beyond that point then you lose accuracy. We also had to make sure to place any writing on the side of the tubes we used for our PCR reaction mix that way we wouldn't run any risks of losing our labels during the heating process of our PCR. We did have about 20uL of leftover PCR reaction mix since we only took about 80uL of our 100uL volume.

Fluorimeter Procedure

Imaging set-up

  1. Place the fluorimeter on top of a two plastic boxes provided in BME lab.
  2. Set up the phone cradle 6 cm away from the fluorimeter.
  3. Check to make sure that the camera is at a 90 degree angle from where the drops will be placed.
  4. Disable the flash on the Camera.


Placing Samples onto the Fluorimeter

  1. Set a pipettor to 80 microliters.
  2. Place 80 microliters of SYBR GREEN I inbetween the two middle dots of the first two rows.
  3. Add 80 microliters of either a calf thymus dilution, or PCR sample to the drop made in step 2.
  4. Once the sample is captured remove the dot with a pipettor and discard the waste.
  5. Shift the slide back two rows for the next sample and repeat from step 2.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

Pcr5g4.png 5 μg/mL sample droplet with circle on ImageJ.

Pcr05g4.png 0.5 μg/mL sample droplet with circle on ImageJ

Pcr0g4.png Zero DNA sample droplet with circle on ImageJ


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 MEAN Standard Deviation
Image 1 Image 2 Image 3
5 2.5 C-1 7732532 7789265 9774402 8432066.333 1162842.827
2 1 C-2 8394697 8119911 7835912 8116840 279405.158
1 0.5 C-3 6060344 6465395 6261127 6262288.667 202527.9987
0.5 0.25 C-4 4061005 2426715 4429557 3639092.333 1065998.003
0.25 0.125 C-5 3336390 3765723 3291584 3464565.667 261770.3172
0 0 C-6 3596959 3318023 3348723 3421235 152953.6388


Calibration curves
Plot1g4.png Plot2g4.png


Images of Our PCR Negative and Positive Controls

Pcrpg4.png Positive control PCR sample droplet on ImageJ.

Pcrng4.png Negative control PCR sample droplet on ImageJ.

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)
G4 + 8533188 1.058305385 12 12.69966462
G4 - 2963613 -0.01276673077 12 -0.1532007692
G4 1-1 4587493 0.2995178846 12 3.594214615
G4 1-2 3682424 0.1254661538 12 1.505593846
G4 1-3 2865087 -0.03171403846 12 -0.3805684615
G4 2-1 4448538 0.2727957692 12 3.273549231
G4 2-2 4067112 0.1994446154 12 2.393335385
G4 2-3 4012010 0.1888480769 12 2.266176923


PCR Results: Summary

  • Our positive control PCR result was 12.69966 μg/mL
  • Our negative control PCR result was -0.15320 μg/mL


Observed results

  • Patient 57569 : The droplet showed to be clear after the SYBR Green was added and there was no green light detected when positioned the the LED light beam. Therefore, we can say that our patient is negative for the SNP disease. The PCR results for this patient were closer to the negative control when compared to both the positive and negative controls.
  • Patient 98829 : The droplet showed to be clear after the SYBR Green was added and there was no green light detected when positioned the the LED light beam. Therefore, we can say that our patient is negative for the SNP disease. The PCR results for this patient were also closer to the negative control when compared to both the positive and negative controls.


Conclusions

  • Patient 57569 : This patient tested Negative for the single-nucleotide polymorphism (SNP) disease.
  • Patient 98829 : This patient tested Negative for the single-nucleotide polymorphism (SNP) disease.