The materials used in the lab included the following: a red solo cup (to dispose of used tips), pipette tips, rack, micropipettor, 8 PCR tubes, PCR reaction mix, DNA primer mix, PCR machine, lab coat, and gloves.
The steps of the experiment were followed below:
1. The empty PCR tubes were labelled on the sides like so:
positive control: G8 + (this was a different label than the one in lab A)
negative control: G8 = (this was a different label than the one in lab A)
patient 1, replicate 1: G8 1-1
patient 1, replicate 2: G8 1-2
patient 1, replicate 3: G8 1-3
patient 2, replicate 1: G8 2-1
patient 2, replicate 2: G8 2-2
patient 2, replicate 3: G8 2-3
2. The PCR tubes were cut, so that there were 2 strips of 4 and placed in the rack.
3. The micropipettor was set to 50 μL. Then, using the correct pipetting technique (where by a the pipette was pressed onto the tip to attach the tip, the pipette was pressed to the first stop, inserted into the PCR reaction mix, the plunger was slowly released, there was a pause for 1-2 seconds, and the tip was lifted straight out of the liquid), 50 μL of PCR reaction mix was obtained. Then, the PCR reaction mix was transferred into an empty PCR tube using the correct expulsion technique (the tip was placed in the delivery tube, the plunger was pressed to the second stop, there was a 1-2 second pause, the tip was lifted out of the tube, the plunger was slowly released, and the used tip was ejected). There was no confusion between the first and second stop on the micropipettor. No liquid was left in the PCR reaction mix tubes. When this step was repeated for the other tubes, where 50 μL of PCR reaction mix were transferred into each of the empty tubes, there was an equal volume in each of the tubes.
4. Then, using the same technique, 50 μL of the correct DNA/ primer mix was added to each of the tubes, for a total volume of 100 μL in each tube. In the same way as the pervious step, no extra liquid was left over in the DNA/primer mix tubes, and al the delivery tubes had equal volumes.
5. The lids on the PCR tubes were closed and the tubes were placed in the PCR machine.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S5
Flash: off
ISO setting: 800
White Balance: Auto
Exposure: +2 (highest setting)
Saturation: auto
Contrast: auto
Camera set-up
Distance between the smart phone cradle and drop = 6 cm
Placing Samples onto the Fluorimeter
First, place 80 uL of SYBR Green on the rough side of the slide, then place 80 uL of the PCR sample on top of the SYBR Green.
Move the slide to focus the blue light as it goes through the 180 uL of the SYBR Green/PCR solution.
Ensure the distance between the phone and blue light is adequate enough to take a clear picture.
Turn on the timer and lower the light box flap to take the best picture possible.
After opening the flap, remove the sample with a micropipetter and repeat steps for the next sample.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
(From left to right: high, zero, low)
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
(From left to right: Negative and Positive)
PCR Results: PCR concentrations solved
Note: The above table shows negative values for the concentration of DNA in the samples. The group is aware that this is not possible, and is due to a skewed relationship in the linear calibration from table 2. These negative values should actually be recorded as zero. However, for the sake of showing that the calculations were done correctly, they remain negative in the above graph.
PCR Results: Summary
Our positive control PCR result was 85.46982 μg/mL
Our negative control PCR result was 17.259152 μg/mL
Observed results
Patient 61878 : The drop is a clear colorless liquid when tested using the flurometer, no pigments of green are expressed in the image. A negative value for the initial concentration of the first two samples was obtained (to be understood as zero), and the final sample had a concentration of 7.004644 μg/mL.
Patient 40819 : The drop is a clear colorless liquid when tested using the flurometer, no pigments of green are expressed in the image. A negative value for the initial concentration of the first two samples was obtained (to be understood as zero), and the final sample had a concentration of 12.616962 μg/mL.
Conclusions
Patient 61878 : This patient's results for the concentration of DNA were all less than both the positive and negative controls. Therefore, this patient is negative and will not express the single nucleotide polymorphism.
Patient 40819 : This patient's results for the concentration of DNA were all less than both the positive and negative controls. This patient is negative and therefore will not express the single nucleotide polymorphism.
BME 100 Fall 2015
