BME100 f2018:Group2 T0800 L5

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OUR TEAM

Name: Kayla Charles

Name: Youssef Serag

Name: Itzel Pineda

Name: Tim Sayre

Name: Ami Asodji

LAB 5 WRITE-UP

PCR Reaction Report

Our team had a positive experience with the pipetting. We all shared the task, so we were all able to practice the technique. Every group member took the prelab quiz and watched the videos on pipetting so it made the task fairly simple. All of our team members understood that the first stop on the pipettor was for taking liquid out and the second stop was for placing liquid into the tubes. We also knew to watch out for bubbles when transferring liquid and not contaminate the pipettor by laying it down. The final reactions did not have the exact same amount of liquid. This is because it is impossible to gather exactly the same amount of liquid everytime. There are so many things that could have affected this such as different placements in the tubes. We were also switching team members and everyone has their own way of pipetting. There was a little bit of liquid left in the DNA samples and the PCR reaction mix. In the end we did not have to change our labeling scheme.

Fluorimeter Procedure

Imaging Set-Up:

SMARTPHONE 1: Iphone 8

1. Place the fluorimeter on an elevated surface to align it with the phone camera.
2. Place the phone on the stand that was collected at the beginning of the lab, and make sure that the camera is set up in front of the place where the droplet is going to lay.
3. Move the phone as close as possible to the fluorimeter setup.
4. Doing so will allow the camera to get the clearest pictures.
5. Line up the hole where the blue LED light comes out from with the position the drop is supposed to be placed upon.
6. Once the drop is placed on the slide, take 3 clear pictures of the droplet.
7. After taking pictures of the droplet, remove the box, and repeat the previous steps for all the samples.

Placing Samples onto the Fluorimeter:

1. Place the first droplet in the middle of the first 2 rows of the slide.
2. Make sure that the blue LED light is placed between the first 2 rows of the slide. If it is placed correctly, it should go through the water droplet.
3. Using a new pipette tip, suck up 80 microliters of the SYBR GREEN and place on top of the first droplet that is already on the slide.
4. Make sure to eject the tip that was used into the waste cup.
5. After combining the 2 droplets, a huge drop should form on the slide.
6. Remove the used tip into the waste bin.
7. Set the micropipette to 160 microliters and pipette the big droplet, remove the tip from the micropipette into the waste bin.
8. Move the slide to the next 2 rows, and make sure that the blue LED light to shine between them.
9. Repeat the previous steps for each PCR tube, and complete taking pictures of all the droplets.

Data Collection and Analysis:

Images of High, Low, and Zero Calf Thymus DNA

5 μg/mL

0 μg/mL

.5 μg/mL

Calibrator Mean Values:

5 μg/mL

Calibration curves:

Images of Our PCR Negative and Positive Controls:

Positive 5 μg/mL

Negative 0 μg/mL

PCR Results: PCR concentrations solved:

PCR Results: Summary

• Our positive control PCR result was 5 μg/mL
• Our negative control PCR result was 0 μg/mL

Observed results

• Patient 55505: The pictures of patient 55505 looked similar to the negative control. The negative control was almost gray with no color and this is how patient 55505's pictures looked. From looking at the results we determined patient 55505 was negative.
• Patient 86299: The positive control picture was green and when we compared it to patient 86299's pictures we can see a similarity. Patient 86299's pictures were similar to the positive control so this means that patient 86299 was positive.

Conclusions

• Patient 55505: Patient 55505 is negative because after comparing the results to both controls we can see that it looks similar to the negative control. There was no green color in the sample. This means that patient 55505 does not have the disease.
• Patient 86299: Patient 86299 is positive because after comparing the results to both controls we can see that it looks similar to the positive control. The sample was green. This means that patient 82699 has the disease.