BME100 s2017:Group4 W1030AM L5

BME 100 Spring 2017	Home People Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3 Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6 Course Logistics For Instructors Photos Wiki Editing Help
OUR TEAM Name: Chase Wallace Name: Lillie Robinson Name: Nick Holmes Name: Kyle Xue Name: Zoe Bargas Brenna Hickey LAB 5 WRITE-UP PCR Reaction Report Overall, our team had a very positive experience with pipetting the samples to set up the reaction. This comes as a result of what we learned from reading the pre-lab as well as teamwork during pipetting. The pre-lab reading adequately prepared us in how to use the micropipettes because it taught us how to set them to desired volumes as well as how to not go over or under the set volume by incorrectly using the first and second stop. Due to our preparation, the final reactions all ended up with the same amount of liquid and there was none left over in the DNA samples and the PCR reaction. Throughout pipetting, we experienced no error large enough to force us to change our labeling scheme. However, sometimes air would accidentally get sucked in and form air bubbles. Although the air bubbles did cost us some time, they were easy to deal with as we simply re-pipetted the desired volume. In addition, we had one person focusing on micropipetting while two others supported that person by managing which tubes they were drawing liquid from and checking the set volume at certain intervals. Teamwork and preparation allowed us to have efficient micropipetting. Fluorimeter Procedure Imaging set-up Camera Procedure: First, we determined and what model of camera would be used to take photos. Once the camera was picked, the flash on that phone was turned off. Then we set the white balance to auto, the ISO, the exposure, and saturation to the highest setting, and the contrast to the lowest. Once the technical settings were completed, we set the camera in the dock, and moved the fluorimeter to the appropriate height to take a picture. We made sure the camera was in focus between takes and adjusted it such that the camera was level with the drop.When the phone was determined to be in the ready position, we turned on the timer, closed the lightbox shade, and let the camera take its 3 pictures. Placing Samples onto the Fluorimeter Set up the fluorimeter and turn it on Place the slide on the fluorimeter with the “smooth” side down Place the first drop of 80 uL on the first two clear circles in the middle of the slide Place the second drop on top of the first one Adjust the slide so the light illuminates the drop and focuses on the droplet Cover fluorimeter with lightbox, then proceed to take pictures Remove the 160 uL drop from the slide and discard liquid into liquid waste container Move the slide into the next position, so that the light is shining in between the next two middle circles Repeat steps until all samples have been completed Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Zero Calf Thymus DNA Low Calf Thymus DNA High Calf Thymus DNA Calibrator Mean Values Calibration curves Images of Our PCR Negative and Positive Controls Negative Control Positive Control PCR Results: PCR concentrations solved PCR Results: Summary Our positive control PCR result was 14.05 μg/mL Our negative control PCR result was -0.31 μg/mL Observed results Patient 50919 (Denoted as the G4 1- # tubes) : From a qualitative perspective, the pictures of patient 50919's pcr products seemed to contain no significantly visible amounts of green. The only dominant color was the blue light of the fluorimeter shining through it. The quantitative results also support the qualitative observations because all 3 replicates of this patient's DNA had concentrations of approximately -0.2, -0.01, and -0.5 micrograms per milliliter. These very slight negative values(which are close to 0) closely resemble the slight negative value of the negative control. Quantitatively, there seems to be effectively zero pcr product concentration in this patient's pcr tubes. Patient 67309 (Denoted as the G4 2- # tubes) : From a qualitative perspective, the pictures of patient 67309's pcr products seemed to contain significantly visible amounts of green coloration. Green is clearly visible when the blue light of the fluorimeter shines through the droplets of this patient's pcr product mixed with the SYBR Green I. The quantitative results seem to support the qualitative observations because the 3 replicates of this patient's DNA had concentrations of approximately 12.9, 13.4, and 14 ug/mL. These values of initial pcr product concentration are very close to the positive control's concentration of about 14 ug/mL. There seems to be a significant amount of pcr product in patient 67309's pcr tubes relative to the positive and negative controls. Conclusions Patient 50919 : Based on how close the pcr product concentration values of the 3 replicates of this patient's DNA were to the negative control, it is reasonable to conclude that patient 50919 is negative for the disease SNP. Theoretically, the negative control concentration should be 0 because the desired DNA segment is not present in the negative sample, but within the context of error introduced by imperfect calibration curves, the concentrations of the negative control and patient 50919 end up being slightly negative. Patient 67309 : Based on how close the pcr product concentration values of the 3 replicates of this patient's DNA were to the positive control, it is reasonable to conclude 67309 is positive for the disease SNP. Theoretically, the positive control's template DNA possesses the desired segment of replication so the pcr product concentration should be a value greater than 0. Since the value of the pcr product concentrations for the positive control is assumed to be a good gauge for whether or not samples are positive, patient 67309's pcr product concentrations that have close values to the positive control are also deduced to be positive. Gel Electrophoresis Image of gel, test labels, list corresponding to each lane. (from left to right) DNA ladder, G41-1, G41-2, G41-3, G4+, G42-1, G42-2, G42-3, G4- Patient 2’s DNA samples correspond to the positive control indicating that the patient displays the positive characteristics and should be treated. Patient 2 and the positive control each display equal DNA lengths that correspond to approximately 128 bp.