LAB 5 WRITE-UP
PCR Reaction Report
Some group members were already experienced micropipette users, others were not. The pre-lab pipetting exercises definitely helped the beginners understand how to handle one. The 1st and 2nd stop mechanisms were easy to understand in theory, but it took some practice for the beginners to fully grasp. Eventually they understood how the first stop is the seal to extract solutions, and the second is the release. It would have been preferred that the pre-lab was more interactive, similar to sections A and B, but it's underline goal was achieved.
The final reaction did not have all of the liquid, because there was left over liquid in the dna samples tube. However, we did extract the large majority. The full amount was not gathered and extracted due to improper pipetting techniques and air bubbles.
Labeling the tubes was difficult due to their small nature. Our group kept our initial scheme, but simply wrote on the number of the added solution on the label.
- Open the front panel of the lightbox.
- Place the box on the table with an open front panel and a closed roof.
- Place the fluorimeter in the dark depths of the box to avoid light exposure.
- Tuck the glass slide into the fluorimeter with the rough side up.
- Station the smartphone on the cradle more than 4cm away from the fluorimeter.
- Position the camera lens so that it has a leveled view of the drop samples.
Calibrating the Fluorimeter
- Using a micropipette, release a 160µL drop of water (H2O) between the first two clear rows on the glass slide.
- Power the fluorimeter's blue LED light.
- Configure the smartphone with the proper camera settings.
- Position the smartphone on the cradle in a fashion that would allow the camera lens to be leveled with the drop.
- Adjust the distance between the cradle and the drop to allow a clear picture to be taken, no more than 4cm away.
- Record the exact distance between the cradle and the drop.
Placing Samples onto the Fluorimeter
- Extract 80µL of the SYBR Green I solution with a micropipette.
- Release 80µL of SYBR Green I solution from the micropipette between the first 2 clear circles of the glass slide.
- Extract 80µL of the 0.25 Calf Thymus DNA solution with a micropipette.
- Release 80µL of Calf Thymus DNA solution from the micropipette onto the SYBR Green I drop, creating a single 160µL drop containing both solutions.
- Accommodate the glass slide to allow the center of the drop to light up while emitting a beam of light on the other side.
- Verify that the camera is focused on the 160µL drop, record the distance between the camera and drop, make sure that they are more than 4cm apart.
- Enclose the fluorimeter and smartphone within the lightbox, make sure that the front flap of the box is open.
- Double check that the camera is focused on the drop.
- Set up the timer on the camera so that it snaps a picture of the drop after the fluorimeter and smartphone are fully enveloped inside the lightbox with the front flap sealed.
- Open the box and retrieve the 160µL drop from the glass slide with a micropipette, then release it in the liquid waste container.
- Remove the glass slide from the fluorimeter, and dispose of it in the designated waste location.
- Insert a new slide into the fluorimeter
- Repeat steps 1-12 four times for each remaining slide center, and each remaining Calf Thymus DNA
Preparing the PCR products
- Retrieve the group's PCR samples from the TA.
- Label the buffers witht he red dots so that they match each PCR sample label.
- With a micropipette, put 100µL of the PCR solution into the buffer with the correct label.
- Enclose the solution product
- repeat for the remaining 7 PCR reaction tubes.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
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 316.4614763 μg/mL
- Our negative control PCR result was 140.0889613 μg/mL
- Patient 45622 : For this patient there seemed to be very little green coloration within the pictures. The highest μg/mL concentration was 199.906 μg/mL. This concentration was hardly comparable to the 316.4614763 μg/mL positive control. Furthermore, it should be noted that our sample of G11 1-1 was contaminated therefore we were not able to use that as a viable sample for observation.
- Patient 73067 : For this patient, we were able to observe all the samples given to us; however, the concentrations found in this patient were much lower than even the negative control ranging from 83.99 to 112.52 μg/mL.
- Patient 45622 : This concentration was hardly comparable to the 316.4614763 μg/mL positive control. Furthermore, it should be noted that our sample of G11 1-1 was contaminated therefore we were not able to use that as a viable sample for observation. This is indicative of there being a possibility of error. However, we are drawing the conclusion that this might have a negative result as the current positive and negative median is 228.27 μg/mL and the 199.906 μg/mL does not meet the cutoff.
- Patient 73067 : For this patient, the concentration ranging from 83.99 to 112.52 μg/mL is far lower than patient 45622 as well as the negative control. This observation, while staying consistent with the first, falls below the median of 228.27 μg/mL therefore possesses a negative result. However, this patient possesses a MORE negative result than the first.