BME100 f2018:Group2 T1030 L5

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OUR TEAM

Name: Daniella Pautz
Name: Amy Floyd
Name: Ally Spohn
Name: Jackson Gates
Name: Nick Hagan


LAB 5 WRITE-UP

PCR Reaction Report

PCR Reaction

For the PCR Reactions Lab our group arrived in class one member short, Jackson was missing.
First, Nick collected all the dry and consumable materials required for the lab. These included: empty PCR tubes, scissors, a sharpie, disposable pipette tips, a cup for discarding the disposable tips, a micropipettor, and a tube holding container.
Second, Amy labelled all the eight tubes with the correct labels on the side as listed in the chart below.
Third, Nick collected all the wet materials required for this lab. These included: both the PCR reaction mix, our patient specific (15385, 95785) DNA primer sample mixes, and the positive/negative controls. During this time and the next following steps, Ally and Daniella started working on the lab 5 write-up and his lab report.
Fourth, Nick and Amy took turns distributing all the wet materials into the corresponding labelled PCR tubes. We encountered difficulties with correctly using the micropipettor. For almost every sample we had to push past the first stop in order to get any liquid from the primer mixes into the pipette tip. Because of this, we certainly have some variation in sample size between the tested tubes. However, we tried our best to make them all the same. Also, despite the prelab reading we were still a little confused about what primer was going where. Perhaps there should be an additional step created that would specify each mixture.
Fifth, Amy took the prepared PCR tubes to a thermocycler machine. We were paired with group 15, with whom we jointly entered all the correct temperatures, cycles, time intervals, and steps into the OpenPCR program. Once entered, we closed and tightened the lid before pressing start. These specifications are also included in the second chart below.
Sixth, all group members contributed in cleaning up the mess and leftover materials from the lab.
Lastly, we as a group sat during the leftover allotted lab time and wrote this lab report.


PCR Reaction Sample List

Tube Label PCR Reaction Sample Patient ID
G2 + Positive control 15385
G2 - Negative control 95785
G2 1-1 Patient 1, replicate 1 15385
G2 1-2 Patient 1, replicate 2 15385
G2 1-3 Patient 1, replicate 3 15385
G2 2-1 Patient 2, replicate 1 95785
G2 2-2 Patient 2, replicate 2 95785
G2 2-3 Patient 2, replicate 3 95785

Thermocycler Specifications

INITIAL STEP: 95°C for 2 minutes:
Denature at 95°C for 30 seconds:
Anneal at 57°C for 30 seconds:
Extend at 72°C for seconds:
FINAL STEP: 72°C for 2 minutes:
FINAL HOLD: 4°C

Fluorimeter Procedure

Fluorimeter

Imaging set-up

To set up the device, our group used the instructions listed on PreLab Orientation document for calibration. First, we turned on the fluorimter and placed it on the table. Then, wearing gloves, we determined which side of the slide was the smooth side. We placed the slide in the fluorimeter with the smooth side down. Next, we adjusted the height of the fluriometer to get a camera view of the slide. The phone was positioned a distance of 4 cm from the fluorimter. A timer was set for 3 seconds on the iphone 7. From there, we began the experiment.


Placing Samples onto the Fluorimeter

Materials:

  1. 8 tubes marked with red dots: 500 microliters of Buffer
  2. 2 tubes labeled with blue "S": 1,000 microliters of SYBR Green 1 Solution
  3. 1 tube labeled H2O: 1,000 microliters of water at pH=8
  4. 5 tubes labeled 0.25, 0.5, 1, 2, and 5: double stranded Calf Thymus DNA

Procedure

  1. With the pipettor, place a 80 microliter drop of SYBR GREEN I onto the middle of the first two dotted rows of the slide
  2. Add 80 microliters of the calf thymus solution labeled 5 to the first drop of SYBR
  3. Turn the blue LED light on and make sure it is focused through the drop
  4. Ensure that the phone is still propely ajusted from the previous part and that the time is on
  5. Begin to capture images of the slide after the light box is closed with as little light as possible
  6. Remove the box, but do not remove the phone
  7. Remove the 160 microliter solution drop from the slide using the pipettor and dispose into the waste bucket provided
  8. Repeat steps 1-7 previously mentioned for the other calf thymus solutions, 2, 1, 0.5, and 0.25



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

5 micrograms/mL
.5 micrograms/mL
0 micrograms/mL

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 94680 94680 93486 94282 689.3562214
2 1 C-2 64629 63990 64629 64416 368.926822
1 0.5 C-3 55087 55087 57598 55924 1449.726526
0.5 0.25 C-4 27883 26134 25047 26354.66667 1430.819462
0.25 0.125 C-5 17391 17356 16659 17135.33333 412.8877975
0 0 C-6 24988 25735 25698 25473.66667 421.0063341


Calibration curves

G2 dot plot 1.JPG
G2 dot plot 2.JPG


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)"
G2 + 3253959.66 67.11011496 12 805.3213795
G2 1-1 3045359 62.78158201 12 753.3789841
G2 1-2 2803806.33 57.76928391 12 693.2314069
G2 1-3 3421804 70.59294074 12 847.1152888
G2 - 2276133.33 46.81989397 12 561.8387276
G2 2-1 2806993.66 57.83542206 12 694.0250647
G2 2-2 2940361.66 60.60285234 12 727.2342281
G2 2-3 2857547.33 58.8844275 12 706.61313


PCR Results: Summary

  • Our positive control PCR result was 805.3 μg/mL
  • Our negative control PCR result was 561.8 μg/mL


Observed results

  • Patient 15385 : We observed a green fluorescence emitting from each of patient 1's samples. We recorded and calculated PCR product concentrations of 753.4 µg /mL, 693.2 µg /mL, and 847.1 µg /mL for each set of patient 1's samples. These concentrations are around our calculated positive control's threshold and well above our negative control's 561.8 µg /mL.
Patient 15385

  • Patient 95785 : We observed a green fluorescence emitting from each of patient 2's samples as well. We recorded and calculated PCR product concentrations of 694.0 µg /mL, 727.2 µg /mL, and 706.6 µg /mL. Just as with patient 1, the concentrations for patient 2's samples are all well above the negative control's threshold of 561.8 µg /mL.
Patient 95785

Conclusions

  • Patient 15385 :
    We concluded patient 1 has tested positive for the disease. We base this on the bright green fluorescence of each of their samples as well as the calculated PCR product concentrations being above the negative control's threshold of 561.8 561.8 µg /mL.
  • Patient 95785 :
    We have also concluded that patient 2 has tested positive for the disease. We base this on the bright green fluorescence of each of their samples as well as the calculated PCR product concentrations being above the negative control's threshold of 561.8 561.8 µg /mL.