DNA Sample Set-up Procedure
Step 1 Gather all materials
Step 2 Cut the strip of PCR tubes so there are 2 strips of four tubes
Step 3 Label the empty tubes
Step 4 Place tubes in rack
Step 5 Take the tube labeled positive control and pipette 50 μL of PCR reaction mix in the tube
Step 6 Using a new pipette transfer the positive control DNA into the same tube. Mix.
Step 7 Repeat steps 5 & 6 for negative control patient 1 (1-1, 1-2, 1-3) and patient 2 (2-1, 2-2, 2-3)
Step 8 All tubes should contain a 100 μL mixture of DNA/primer mix and PCR mix
Step 9 Close lids on all PCR tubes tightly
Step 10 Place the tubes into the slots in the PCR machine in the heating block (Don't run machine until all the slots are filled)
OpenPCR program
HEATED LID: 100°C
INITIAL STEP: 95°C
NUMBER OF CYCLES: 25 Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
The teams experience with pipetting the samples to set up the reaction was very smooth and accurate. Before beginning the process of mixing agents for the PCR reaction with the DNA samples, the pre-lab work was consulted. Without this additional information contained within the pre-lab there would be a much higher chance of a failed experiment. The measurements and details on how to mix the PCR reaction agents was very helpful. The guided process made it easier to follow the newly learned procedures.
Understanding the difference between the first stop and the second stop on the pipette was a necessity for this lab. The first stop on the pipette sets the measured amount you wish to pull from the liquid when released. The second stop allows for an extra expulsion of air to make sure all liquid gathered during release of the first stop is expelled. The second stop is only to be used once the liquid has been gathered and is needing to be expelled, then the pipette is released to the second stop to ensure all liquid gathered is pushed out.
Since there was no loss of any material during this procedure the liquid remained the same during and after the reaction. The reaction was simply adding the DNA to the mix of polymers and primer. This means that the two liquids were just added together to find the total volume and keep the total volume constant and without change.
In the original DNA and PCR mixes there were still leftover DNA and PCR mix that was unused. The experiment only required 50uL of each for a total of 100uL. This unused liquid was thrown in the trash since it was extra that could have possibly contaminated the other tubes if they were to mix at some point. There were multiple tubes for each reaction, and since there were multiple tubes each designated for a specific reaction there was no cross contamination effecting the other trials. Not all of the DNA or mix was used in every trial to make sure that there was a sufficient amount of both to perform the experiment.
For this experiment the labels of every mixture was the same throughout the experiment. This team was able to make sure that each component was properly transferred into each corresponding tube to make the correct mixture and keep every label consistent.
Fluorimeter Procedure
Imaging set-up
Materials were gathered
The box was set on its side with the flap at the top
A phone was placed in the phone holder
Some boxes were stacked to match the height of the camera on the phone
A clean slide was placed in the slide holder part of the light box and the Blue LED light was turned on
The light box was put on top of the stacked boxes
Placing Samples onto the Fluorimeter
A pipette was used to obtain 80 microlitres of SYBR Green
It was carefully placed in a droplet shape on the rough side of the slide, perpendicular to the light
The pipette tip was properly disposed of, and a new, sterile one was used
80 microlitres of the 0 concentration control (C-6) were put on top of the SYBR droplet
The slid holder was placed on the stacked boxes and slid into the back of the box
The phone was positioned so that the camera had the samples in view
The camera was put on a timer, and the box flap was closed to block outside light
Three pictures were taken of each sample
Observations were recorded
The procedure was repeated until all of the samples were pictured
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was -0.8745636 μg/mL
Our negative control PCR result was 28.16076 μg/mL
Observed results
Patient 65580 :
Quantitative: The initial PCR Product Concentration for this patient was 90.39144 μg/mL
Qualitative: The fluorescent drops were brighter once color channels were split in Image J while other drops did not fluoresce very much of the color green.
Patient 27833 :
Quantitative: The initial PCR Product Concentration for this patient was 67.088916 μg/mL
Qualitative: The fluorescent drops were brighter once color channels were split in image J while other drops did not fluoresce very much of the color green.
Conclusions
Patient 65580 : Patient number 1 (patient ID 65580) had a negative result when compared to the drop fluorimeter positive control test. Whereas the positive control for the test had a visible green glow, the patient's DNA when tested had no color visible. The DNA from the patient matched the negative control where there was also no color present beyond the blue tint from the blue light given off by the machine. Based on the results, it is concluded that patient number 1 (patient ID 65580) does not have the disease and the result of the test is negative. This is because the results matched the negative control test and did not change to exhibit the green glow of the positive control test. This data is also backed up by the matching result of the gel electrophoresis test.
Patient 27833 : Patient number 2 (patient ID 27833), like patient number 1, also had a negative result when compared to the drop fluorimeter positive control test. Whereas the positive control for the test had a visible green glow, the patient's DNA when tested had no color visible. The DNA from the patient matched the negative control where there was also no color present beyond the blue tint from the blue light given off by the machine. Based on the results, it is concluded that patient number 2 (patient ID 27833) does not have the disease and the result of the test is negative. This is because the results matched the negative control test and did not change to exhibit the green glow of the positive control test. This data is also backed up by the matching result of the gel electrophoresis test.
Gel Electrophoresis
Gel Electrophoresis
The image of the gel electrophoresis seen here shows that there were no patients with the disease marker in their DNA. The image (from bottom to top) shows in the first lane (very bottom row) the positive marker, followed by the negative marker in the 2nd row. The very last lane (top row) shows the DNA ladder that was used for comparison.
In the 3rd, 4th and 5th row (from bottom to top) are the DNA PCR samples from patient number 1 (patient ID 65580). As you can see, compared to the positive marker, there is no indicator of a match for the disease.
In the 6th, 7th and 8th rows (from bottom to top) are DNA PCR samples from patient number 2 (patient ID 27833). Just as seen in the first patient, there is no indicator for the disease marker.
The results of the gel electrophoresis match the results from the drop fluorimeter with both tests showing no positive indicators besides the positive control marker.
BME 100 Spring 2017
