BME100 f2015:Group1 1030amL5

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OUR TEAM

Name: Matt Chrest
Role(s)
Name: Lindsey O'Brien
Role(s)
Name: Jacob Aperi
Role(s)
Name: Michael Otis Clyne
Role(s)
Name: Nichole Torgerson
Role(s)
Name: Alarmel Sira
Role(s)


LAB 5 WRITE-UP

PCR Reaction Report

Due to the pre-lab materials, and some of our team member’s previous experience, our team was well informed before the lab of proper pipetting technique. To begin the lab we gathered our materials and properly labeled the sides of the PCR tubes for our group, following our template from part A of the lab. Our tubes were labeled as: G1 +, G1 -, G1 1-1, G1 1-2, G1 1-3, and G1 2-1, G1 2-2, G1 2-3, to show the different trials for each patient, along with our positive and negative controls. Before we began pipetting, we made sure our samples were fully thawed, in order to get correct measurements from the pipetting. Our pipetting was done in a group of two, for continuous quality check. We had some volume issues with our pipetting, and with the assistance of Dr. Haynes and some extra aster mix, these issues were corrected, giving us the proper volume of 50 µL in each PCR tube. After they were all properly pipetted, we programmed the PCR machine in accordance to the Lab Workbook. We then notified a TA, and they helped us load the tubes into the PCR machine, and have it properly programmed.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone (back-up), Samsung Galaxy Note 4 (used)
    • Flash: off
    • ISO setting: No setting, Set to 800
    • White Balance:No setting, Auto
    • Exposure: No setting, +2: highest setting
    • Saturation: No setting, No setting
    • Contrast: No setting, -2: lowest


Camera set-up
We placed the fluorimeter on top of two empty pipette tip trays, and then positioned the camera 8 cm away from the flourimeter, adjusted in place by folded paper, in order to keep the phone straight up so we can take a sideways picture. The camera timer was set at 5 seconds. We marked the location and remeasured the distance when we moved the camera between trials

  • Distance between the smart phone cradle and drop = 8 cm


Placing Samples onto the Fluorimeter

  1. Attach a new pipette tip to the micropipetter, which is set at 80 μL
  2. Add 80 μL of SYBR Green I Solution in a single drop between the middle circles in the first two rows on the rough surface of a new slide
  3. With a new pipette tip, add 80 μL of either the sample or blank onto the previously present drop of SYBR Green I
  4. Align blue light in the fluorimeter with the center of the drop
  5. Place the fluorimeter under an upside down box that blocks out all other light
  6. Place the camera with the above-mentioned settings 8 cm in front of the fluorimeter
  7. Press the capture button, thus activating the timer, and close the flap
  8. Wait for the camera to capture the picture and repeat steps 6-8 for a total of 3 pictures
  9. Remove the slide and discard all material on it
  10. Repeat steps 1-9 for each sample needing testing (in this case, 6 times for calibration and 8 times for PCR reactions)


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High Concentration:

LAB 1030HighConcentrationV2.jpg

Low Concentration:

LAB 1030LowConcentrationV2.jpg

No Concentration:

LAB 1030NoConcentrationV2.jpg

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 11500687 11727326 870850 8032954.333 6203599.372
2 1 C-2 12763552 13260820 10054390 12026254 1725689.599
1 0.5 C-3 9120081 9101978 9790216 9337425 392232.9626
0.5 0.25 C-4 7612011 8115471 8292500 8006660.667 353052.5262
0.25 0.125 C-5 3082232 2782889 3201476 3022199 215654.2139
0 0 C-6 3401514 2545859 2851382 2932918.333 433615.6132


Calibration curves

Lab 1030concentrationgraph1.png

Lab 1030concentrationgraph2.png

Images of Our PCR Negative and Positive Controls

Positive Control:

LAB 1030PositiveV3.jpg

Negative Control:

LAB 1030NegativeV3.jpg

PCR Results: PCR concentrations solved

PCR Product TUBE LABEL RAWINTDEN DROP - BACKGROUND ' ' MEAN Standard Deviation
Image 1 Image 2 Image 3
G1 + 11197613 10004860 9616318 10272930.33 824026.5451
G1 - 2557014 2444811 2511807 10175677.33 56453.00416
G1 1-1 3712743 4079116 4283647 4025168.667 289250.0309
G1 1-2 2879984 3237511 3787064 3301519.667 456915.0508
G1 1-3 3650829 3136043 3450691 3412521 259506.9712
G1 2-1 13790299 15301484 16786776 15292853 1498257.145
G1 2-2 9892540 10560817 9130519 9861292 715660.8277
G1 2-3 11688632 12419617 11616580 11908276.33 444297.0073


PCR Results: Summary

  • Our positive control PCR result was 2.136 μg/mL
  • Our negative control PCR result was 2.087 μg/mL


Observed results

  • Patient 30522 : For our first patient, the images from the fluorimeter gave us all images without the green glow,and after calculating the data, we determined that all of the patient's values were below 2.087
  • Patient 46645 : Our second patient's pictures all appeared a vibrant green, and 2 of the 3 values given were about the positive control limit of 2.136


Conclusions

  • Patient 30522 : All three of the patients trials were below the lower bound of 2.087, and the pictures did not have a green tone, we determined finally that our patient 30522 is negative.
  • Patient 46645 : Our second patient had the majority of his values significantly over the higher bound of 2.136, and in our experiments with the fluorimeter, all of the images appeared to have the green glow. Therefore, with the information, we determined that our patient 46645 is positive.