LAB 5 WRITE-UP
PCR Reaction Report
Fortunately, pipetting was not an issue for our group, because there were several group members who knew how to properly use the micropipette. The pre-lab reading was helpful in setting up the lightbox and smartphone with the stand, however, the instructions then began to seem worded in a convoluted way for rather straightforward procedures. The difference between the first stop and second stop on the micropipette was also not very difficult to understand. The final reactions did have the same of liquid and there was no liquid left in the tubes of the DNA samples and PCR mix. Our group's labeling scheme was not altered.
The smartphone used to capture the images was a Samsung Galaxy S6 and the trials were done on the lab counter tops. Prior to taking any images on the device, the flash was inactivated. The phone was placed on a cradle (as it is referred to in the BME Lab) to capture stable photos. The images were to be taken at the side of the fluorimeter to capture the drops of every solution. The distance between the plastic tray containing the drops, was kept at 8 cm for every trial. During these trial, the device would be placed inside of the light box with the purpose being to eliminate as much light as possible.
Placing Samples onto the Fluorimeter
- A 160 microliter drop of water was placed into the first two rows with the rough side of the slide up, ensuring that the pipetted water would bead up.
- The blue LED of the fluorimeter was turned on.
- A smart phone was placed in the plastic tray at a right angle to the droplet so that it was fully visible.
- The distance of the phone from the droplet was recorded, making sure that it was more than 4 cm away and the picture was not blurry.
- The slide was cleaned.
- 80 microliters of the SYBER GREEN were put onto the slide with an additional 80 microliters of calf thymus (water black) making sure that the solution beaded up.
- The LED was aligned to be focused on the drop and three pictures were taken after the light shield went over the fluorimeter.
- The pictures were checked to make sure they were clear.
- The shield was removed.
- Steps 5 through 9 were repeated replacing the calf thymus with the other solutions.
- Slides were changed out as needed.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
0.5 μg/mL sample
5 μg/mL sample
Calibrator Mean Values
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
- Our positive control PCR result was -4.779795528 μg/mL
- Our negative control PCR result was 31.33498949 μg/mL
- Patient 33805 : This patient has an an average initial concentration of 29.4730 μg/mL. From a qualitative standpoint, patient 1's image did not return much green color. The image shows a relatively transparent drop. The PCR concentration 4.911837 μg/mL. The drop that was analyzed was relatively green with blue towards the top of the drop.
- Patient 18900 : This patient has an average initial concentration of 31.2308 μg/mL. Qualitatively, this patients image almost looks identical to patient 1. This makes sense considering that they have very similar initial concentrations. The drop from patient two was solely blue and somewhat clear in the middle of it.
- Patient 33805 :The PCR concentration (4.911837μg/mL ) of this patient aligns with the positive control PCR result.
- Patient 18900 : The average initial concentration of 31.2308 μg/mL is similar to the negative control PCR result of 31.33498949 μg/mL.