Our teams experience with the pipetting went smoothly. We did not face any problems with the process and were able to set up the reaction well. Yes, the prelab reading and videos did help as they took us step by step through the pipetting process which helped us greatly. We understood the difference between the first and second stop of the pipettor. The first stop is used when extracting the primer and the second stop is used when ejecting the primer into the tube. The final reactions had the same amount of liquid and there was no remaining liquid left in the tubes of the DNA samples and PCR reaction liquid. Finally, we did not have to change our labeling scheme, as they proved to suffice.
Fluorimeter Procedure
Imaging set-up
Our group used a Samsung S8 to take our photos.
IF ABLE use camera menu to
inactivate flash (required for photos)
set ISO to 800 (or higher)
set white balance to auto
set exposure to highest setting
set saturation to highest setting
set contrast to lowest setting
Step 1: Obtain a tray of sample tubes from instructor
Step 2: Check contents on rack
It should contain
8 tubes marked with red dots (these contain 500 microliters of buffer)
2 tubes labeled with a blue "S" (these contain 1,000 microliters of SYBR Green solution)
1 tube labeled H2O (this contains 1,000 microliters of water at pH=8)
Step 3: Place 160 microliter drop of H2O in the middle of the first 2 rows using the pipettor.
Step 4: Turn on the excitation light using the switch for the Blue LED.
Step 5: Turn on the camera of smart phone and check settings (described above)
Step 6: Place smart phone on cradle at right angle from the slide. Adjust the height of the fluorimeter using the plastic trays so the camera takes a sideways picture of the drop.
Step 7: Adjust distance between smartphone on its cradle and the first two rows so it is as close as possible without blurring the image. It should be AT LEAST 4 cm away from drop.
Step 8: Record distance between smart phone and drop using a ruler. Record distance in centimeters.
Placing Samples onto the Fluorimeter
Step one, place 80 microliters of SYBR green I in the middle of first two rows using the pipettor. Add 80 microliters of one of the calf thymus solutions listed above.
Step two, Align the drop by moving the slide so the blue LED is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.
Step three, Use timer on camera so you can take a picture after covering the fluorimeter and camera with the light box. Try to minimize the amount of light coming in.
Step four, Take 3 images, check to make sure drop is focused.
Step five, Remove the box, DO NOT MOVE SMARTPHONE. Check distance of phone with the ruler to ensure it hasn't moved.
Step six, Used pipettor to remove the 160 microliter drop from the surface and move the slide to the next position.
Step seven, Repeat steps 1-6 for other concentrations of calf thymus DNA.
For good data analysis, 3 independent images must be taken of each DNA concentration.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Zero Calf Thymus DNA0.5 Calf Thymus DNA5 Calf Thymus DNA
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative ControlPositive Control
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was 63.794 μg/mL
Our negative control PCR result was 16.953438 μg/mL
Observed results
Patient 1 : The images for patient one were fairly consistent. All images were variations of blue in color and similar in size and shape. None of these images presented any green color in the drops. The concentrations of patient one's samples proved to be consistent with an average value of 18.3 μg/mL. The initial concentrations were 23.5, 13.56, 17.88 across the three samples.
Patient 2 : The images for patient two revealed different levels of green throughout the drop. Sample 2-3 had the most green with majority of the drop being green, sample 2-1 had green color in the bottom of the drop, and sample 2-2 had minimal green color. All drops were similar in size and shape. The initial concentrations were high compared to those of patient one with values of 25.9, 34.4, 63.8 μg/mL.
Conclusions
Patient 1 : After reviewing the results, we have concluded patient one to be negative. With calculations, the values of the initial PCR concentration for patient one prove to be the most similar to the negative control. The average of the three samples for patient one was 18.3, and the negative control value was 16.95. Furthermore, the images of patient one were all blue and revealed no green color.
Patient 2 : After reviewing the results, we have concluded patient two to be positive. With calculations, patient two proved to be more similar to the positive control. Additionally, patient two's images had areas of green color further proving it to have results similar to the positive control.
BME 100 Spring 2018
