This experiment was fairly simple to complete. For this experiment, the prelab did prove itself to be useful. Having prior knowledge about the lab is essential in order to have a smooth running of the lab. The first and second stops on the pipettors are very easy to tell the difference between being that the first has more give after the initial pressure is given and after the second stop the button cannot go down any further. In the case of an unsteady hand, it was found helpful to use a finger from the idle hand and apply a small amount of pressure to the side of the pipette (close to the tip) to prevent further shaking. We made sure to dispose the tip after each transfer. This provided extra prevention of any possible contamination while also allowing us to become more familiar with the use of tips when handling a pipette. After transferring the samples, each tube appeared to have the same volume and every sample tube and PCR reaction tube was left nearly empty (there was some residual liquid but hardly enough to be significant). It can be assumed from these observations that we transferred the volumes correctly. Our labeling scheme did not have to be altered in any way after creating the first key. After closing the tubes and properly cleaning our lab area, we brought the PCR tubes to one of the TAs helping in the lab and she helped us find an open thermal cycling machine to use for our samples. This concluded our first PCR experience.
Fluorimeter Procedure
Imaging set-up
Placed the smartphone on the cradle at a right angle from the slide. We adjusted the height of the fluorimeter using plastic trays so it can get a clear view of the blue LED light hitting the drop.
Placing Samples onto the Fluorimeter
In order to dilute each sample, insert each patient samples to the tubes with red dots containing 500microliters of buffer
Next set up the box needed in order start the fluorimeter.
Use a pipette to take 160 microliters of water and put it between the first 2 rows between the slide)
turn the excitation light on the side of the black small box.
Use the plastic trays given to adjust the height of the small box.
Using the cradle, place the smartphone in front of the small box at a right angle
Keep at least 4 cm distance between the cradle and drop on the slide so the image does not turn out blurry.
Using a ruler, record the distance on the table"
Avoid changing distance in order to prevent errors in measurements of the light collected in the ImageJ program
Using the pipette add 80 microliters of the SYBR GREEN solution. (Remove tip from pipette each time sample is placed)
Align the slide according to the light.
Set the camera timer on the phone. Close the box as the timer starts to avoid extra lights
If adjustment is needed, remove the box ONLY to adjust the slides. Avoid moving other materials like phone etc.
Using the pipette, remove 160 microliters from the slide and move to the next column in the slide.
Repeat steps 10-14 for each concentration
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample:
0.5 μg/mL sample:
Zero μg/mL sample:
Calibrator Mean Values
Calibration curves Images of Our PCR Negative and Positive Controls
Positive control sample:
Negative control sample:
PCR Results: PCR concentrations solved
TABLE GOES HERE
PCR Results: Summary
Our positive control PCR result was 193.74 μg/mL
Our negative control PCR result was 328.87 μg/mL
Observed results
Patient 22111 : Results were 115.29μg/mL, 189.89μg/mL, and 260.67μg/mL. The solutions glowed bright green. As the solution went from sample 1 to 3, it got greener. 1-3 was the brightest of the three.
Patient 14162 : Results were 195.42μg/mL, 270.78μg/mL, and 195.68μg/mL The solutions had a strong glow except for 2-3. 2-2 was the brightest of the three.
Conclusions
Patient 22111 : This patient seems to be closer to the positive control. Our data for the positive and negative controlled got skewed and there was some experimental error. With the calculations that were found, the patient is closer to being positive. The 1-2 had a value of 189.9 μg/mL which is a 3.84 μg/mL difference from the positive.
Patient 14162 : The results for patient two were positive because 2-1 and 2-3 both show signs of being positive. The difference between 2-1 and the positive control is only 1.68μg/mL and the difference between 2-3 and the positive control is 1.94μg/mL.
BME 100 Fall 2018
Images of Our PCR Negative and Positive Controls
