Overall our team's pipette experience proved to be quite successful and accurate, which could be attributed to the pre-lab reading and in class demonstrations. Many of us had never used a micro pipette before this lab. However, after the demonstrations we found that the process was rather simplistic and very efficient. The first level is used for the purpose of gathering a solution into the micro pipette, while the second level is to completely expel the solution into a desired location. After we completed the micro pipette procedure of the lab, we found that for the most part all of the tubes with the DNA sample and the PCR reaction solution had almost identical levels of the solutions across all the tubes. Also, the tubes that were comprised of this DNA sample and PCR reaction did not have to be labeled differently than was already suggested by the lab procedure, which allowed our tunes to be organized and easy to identify. All in all, we found this process to be a beneficial experience and introduction to the usage of micro pipettes.
Fluorimeter Procedure
Imaging set-up
In order to capture the images from the fluorimeter, we created an enclosed box to perform the image capturing. The box contained the fluorimeter which was set up on top of an object in order to make it lens-level with the phone camera that was used to capture the images. The phone was placed on a dock to increase stability and quality of the photo, which in turn resulted in more accurate data. The box was black and covered the other components in effort to produce a dark environment for the most successful results when viewing the samples under the fluorimeter light since the SYBR GREEN 1 solution used to identify positive DNA samples is light sensitive.
Placing Samples onto the Fluorimeter
First things first, make sure to put on a lab coat and gloves for protection.
Obtain a tray of sample tubes (8 tubes marked with red dots for the buffer, 2 tubes marked with a "S" for the SYBR Green 1 solution, 1 tube labeled H2O for water, and 5 tubes labeled 0.25, 0.5, 1, 2, 5 which have double stranded calf thymus DNA within the tubes).
Put the fluorimeter on the table and turn on the excitation light.
Grab a slide and feel for the smooth side, place this side face down onto the fluorimeter.
Turn on the smartphone that is going to be used for capturing the images of the samples, amke sure to make any adjustments necessary to improve picture quality.
Place the phone on a cradle to keep the device stable at a right angle to the slide. Make sure to adjust the height of the fluorimeter with plastic trays so that the phone is taking a picture of the droplet sample from a sideways viewpoint.
Once the height has been properly adjusted, move the phone, which is held by the cradle, as close to the slide as possible. However, do not compromise the quality of the picture in order to get close to the sample. Start from as close the slide as possible and slowly move away until the picture quality is no longer blurry (record this distance).
Then, place a drop (80 microliters) of SYBR Green 1 solution using a pipettor onto the first two rows of the slide.
Add an additional 80 microliters (using a new pipette tip) of one of the calf thymus solutions onto the same location of the slide of the SYBR Green 1 solution. The two samples should cluster together so that there is only one 160 microliter drop (sample) on the slide.
Align the drop so that the light from the fluorimeter is cast directly onto the center of the drop.
After covering the fluorimeter system with a box to limit the exposure to light, use the timer of the smartphone to take a picture after three seconds of clicking the button to take picture. The three seconds will allow time for the box to be placed over the fluorimeter to keep light from entering the system
Repeat the picture taking process two more times so that a total of three pictures are taken for each sample.
Remove the slide from the fluorimeter, making sure not to move the phone.
Repeat steps 4-13 for each sample so that in the end there should be a total of 24 pictures (3 pictures for each of 8 samples)
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
1) 5 μg/mL sample
2) 0.5 μg/mL sample
3) zero DNA sample
Calibrator Mean Values
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive
Negative
PCR Results: PCR concentrations solved
PCR Results: Summary
Our positive control PCR result was -114.7378044 μg/mL
Our negative control PCR result was -129.163056 μg/mL
Observed results
Patient 26464 : The drops of this patient looked clear with the dark blue light encompassing the whole drop. The initial PCR product concentrations of this patient are -124.484256, -140.818692, and -94.2473676 μg/mL, which are lower and more similar to the negative control.
Patient 35032 : The drops of this patient also looked clear with the dark blue light surrounding, however, there is a slight bright white light in the center of the drops. The initial PCR product concentrations of this patient are -109.2275844, -91.5365796, and -95.8778604 μg/mL, which are higher than patient 26464 and more similar to the positive control.
Conclusions
Patient 26464 : The three replicates of this patient's initial PCR product concentration are closer in proximity with the negative control PCR results, therefore, this patient is negative for the disease marker.
Patient 35032 : On the other hand, this patient is positive for the disease marker, because the three replicates of this patient's initial PCR product concentration are closer in proximity with the positive control PCR results.