Name: Christian Sulit
Name: Scott Colebeck
Name: Valerie Cortez
Name: Samson Nguyen
LAB 5 WRITE-UP
PCR Reaction Report
Our team had a very successful experience pipetting the samples to set up the reaction. The pre-lab reading and its diagrams helped a lot with both understanding what we were doing with this process and how to actually perform the process. We understood that the first stop on the pipettor is used to draw up liquid, whereas the second stop is used to expel the liquid. The final reactions ended up having the same amount of liquid as before the reaction. No liquid was left in any of the tubes containing DNA samples or PCR reaction mix. We did not have to change our labeling scheme because the way in which we labeled our test tubes was consistent throughout the lab.
We first propped up our smartphone vertically using the holder provided for us, setting the camera to a 3-second timer. Using some notebooks, we then created an elevated platform upon which the fluorimeter could rest. We then measured and recorded the distance from the fluorimeter to the camera. Next, weplaced one of the slides, with the smooth face down, into the fluorimeter, making sure that the section we would be using lined up with the light being projected. After placing the SYBR green sample and respective DNA sample on the slide, we activated the timer on the phone’s camera and covered the apparatus with the box provided for us to take the picture with a dark background.
Placing Samples onto the Fluorimeter
- Make sure that the slide placed in the fluorimeter has the rough, hydrophobic side facing upwards and that the space between the first 2 clear circles are lined up with the blue light being projected from the fluorimeter.
- Place an 80 µL drop of SYBR GREEN I in the middle of the first two rows of the slide using the pipettor. The drop should look like a beach ball.
- Beginning with a water blank, add 80 µL of one of the provided solutions to the ball of SYBR GREEN I.
- Adjust the slide if necessary to line up the drop with the fluorimeter’s LED light.
- Using the timer function on the smartphone and the box to cover the fluorimeter apparatus, take 3 pictures of the drop. Make sure that the camera is focused on the drop.
- Remove the box, making sure to not move any of the parts of the apparatus in the process. Any accidental movements should be corrected based on the initial distance measurement between the fluorimeter and the smartphone.
- Dispose and replace the slide.
- Repeat Steps 1-7 for the remaining concentrations of calf thymus DNA and patient PCR samples.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
| Final DNA concentration in SYBR Green I solution (µg/mL)
|| Sample Number
|| RAWINTDEN DROP - BACKGROUND: Image 1, Image 2, Image 3
|| Standard Deviation
|| 211051, 207397, 207349
|| 209239, 211391, 217293
|| 218450 160874 210115
|| 205037 217599 148663
|| 191262 180270 148159
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
| PCR Product TUBE LABEL
|| MEAN (of RAWINTDEN DROP - BACKGROUND)
|| PCR Product Concentration (µg /mL)
|| Total Dilution
|| Initial PCR Product Concentration (µg /mL)
| Positive Control
| Negative Control
PCR Results: Summary
- Our positive control PCR result was 275.99 μg/mL
- Our negative control PCR result was 26.71 μg/mL
- Patient 11385 : The image for this particular patient had very little green illumination when the droplets were placed on the fluorimeter. Additional once the image was color separated it was easy to see how transparent the droplet was. The three Initial PCR Product Concentration (µg /mL)for the first patient were as follows: 128.663874 µg /mL, 25.41846012 µg /mL,60.08773961 µg /mL.
- Patient 54376 : The image for this particular patient had very bright green illumination when the droplets were placed on the fluorimeter. Additional once the image was color separated this droplet was much more opaque than that of the other droplet. The three Initial PCR Product Concentration (µg /mL)for the second patient were as follows: 382.0816639 µg /mL, 53.53284182 µg /mL, 83.1875 µg /mL.
- Patient 11385 : The final conclusion for this patient was the they were negative for the disease SNP. This conclusion was based upon both the qualitative and quantitative data. First the images for this first patient very closely resembled that of the negative control. Second the quantitative data with regards to the initial PCR concentration was very similar to that of the negative control
- Patient 54376 :The final conclusion for this patient was the they were in fact positive for the disease SNP. Similar to the first patient, this conclusion was based upon both the qualitative and quantitative data, however the qualitative data proved to be invaluable when coming to this conclusion . The quantitative data for this second patient had similarities with both the positive and negative control, however in one trial is was "overwhelmingly" positive so to speak. The image for this was part of the deciding factors when determining that this patient was positive. The image was a bright green similar to that of the positive control. The image in conjunction with the positive result in the quantitative data help to reach the final conclusion.