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.
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:
8 tubes marked with red dots: 500 microliters of Buffer
2 tubes labeled with blue "S": 1,000 microliters of SYBR Green 1 Solution
1 tube labeled H2O: 1,000 microliters of water at pH=8
5 tubes labeled 0.25, 0.5, 1, 2, and 5: double stranded Calf Thymus DNA
Procedure
With the pipettor, place a 80 microliter drop of SYBR GREEN I onto the middle of the first two dotted rows of the slide
Add 80 microliters of the calf thymus solution labeled 5 to the first drop of SYBR
Turn the blue LED light on and make sure it is focused through the drop
Ensure that the phone is still propely ajusted from the previous part and that the time is on
Begin to capture images of the slide after the light box is closed with as little light as possible
Remove the box, but do not remove the phone
Remove the 160 microliter solution drop from the slide using the pipettor and dispose into the waste bucket provided
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)
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.
BME 100 Fall 2018
