BME100 f2018:Group7 T0800 L5

BME 100 Fall 2018	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: Jeanie Kim Name: Jason Mayo Name: Brodie Clemmer Name: Michelle Rodriguez LAB 5 WRITE-UP PCR Reaction Report We gathered all of the materials in stages, first we were given eight empty PCR tubes and an empty PCR tube rack. Then we were given PCR mix, a positive and negative control, and finally our patient samples. We were advised to handle the materials carefully with disposable gloves and lab coats on. We started pipetting be calibrating the micropipette to 50 microliters. We began placing the PCR mix into each PCR tube, switching people after every two tubes so that each group member could gain experience with performing the experiment. After a little practice and watching the pre-lab video that demonstrated how to pipette, the application was relatively easy. Through brief exposure to using the pipette we were able to distinguish the purposes of the first and second stops on the pipette, which made transferring the liquid easier. We then added the controls and each patient mixture to each PCR tube accordingly. We did not have the same exact amount of liquid present in the final reactions. This is likely due to consistently switching who operated the pipette, because we likely applied different pressures when pressing on the first and second stops. The volume discrepancies could also have occurred due to the placement of the pipette as it gathered the liquid. For instance, we would get slightly different volumes if we placed the pipette tip all the way at the bottom of the tubes, versus somewhere in the middle of the liquid solution. Consequently, there was some liquid left in the tubes due to the different volumes of liquid gathered. We didn't have to change our labeling scheme, but we made sure to follow the procedure by starting with the positive and negative controls, and then going through the 6 replicates of the two patients as found in Part A. Fluorimeter Procedure Imaging set-up Fluorimeter Set-Up The device that was used for this part of the experiment was an iPhone. The first thing we did was to make sure our iPhone was placed in the appropriate position, making sure that the camera lens was consistently lined up with the slide. This ensured having a significant change in the distance between the iPhone cradle and the droplet on the slide. The iPhone was placed on the cradle and a ruler was used to measure the distance. We placed the iPhone cradle 9cm away from the slide, and then checked that the camera was focused on the drop, to prevent blurry pictures. Despite this effort, we had blurry images for the calibration component of data, and so to perform the rest of the calculations as proof of principle, we had to zoom in on the images to get a clearer representation of them for processing in imageJ. Placing Samples onto the Fluorimeter [The first step to placing the samples onto the fluorimeter was using the pipettor to transfer the solutions from the tube to the slide.] [The pipettor collect 80 microliters of SYBR Green and placed a drop of it in the middle of the first two rows of the slide.] [Then the pipettor collected 80 microliters of calf thymus and placed a drop of it on top of the SYBR Green. The result of the drop (as in the shape) should look like an oval.] [Once the photos were taken, the 160 microliters were removed from the slide with a pipettor and placed in the waste container. Then the slide was adjusted to the next set of rows, and the steps were repeated until all the other calf thymus measurements were recorded, making sure to try and avoid cross contamination when at all possible.] Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Calibrator Mean Values Calibration curves Images of Our PCR Negative and Positive Controls PCR Results: PCR concentrations solved Initial Concentration Of 2X Calf Thymus Solution Final Concentration In SYBR Green 1 solution Sample Number RAWINTDEN-Background Mean Standard Deviation Image 1 Image 2 Image 3 5 2.5 C-1 3833486 3740263 9460125 5677958 3275784.34 2 1 C-2 4394411 3878635 4424452 4232499 306823.386 1 .5 C-3 4606213 3957162 5245365 4602913 644107.8389 .5 .25 C-4 5901006 5230118 6023387 5718170 427072.1102 .25 .125 C-5 2628693 2755458 2831652 2738601 102524.1813 0 0 C-6 1759347 1219483 1126960 1368597 341547.2056 PCR Results: Summary Our positive control PCR result was 22.487226 μg/mL Our negative control PCR result was -1.30168 μg/mL Observed results Patient G71 : Patient 1 had a light green due to the PCR reaction having a greater average concentration of 6.213238 μg/mL according to our calibrated imageJ from the calibrated curves from Part 1 Patient G72 : Patient 2 did not appear to have a reaction with the SYBR Green due to having a very low average concentration of 0.85177 μg/mL according to our calibrated imageJ from the calibrated curves from part 1 Conclusions Patient G71 : Based on the data we have collected we have determined Patient 1 to be positive for the this Single Nucleotide Mutation due to the higher PCR product Concentration showing us that the primer was allowed to bind to Patient 1's DNA and complete the PCR process. Patient G72 : Based on the data we have collected we have determined Patient 2 to be negative for the this Single Nucleotide Mutation due to the lower PCR product Concentration showing us that the primer was not allowed to bind to Patient 1's DNA and complete the PCR process. PCR Tube Label Mean Of RAWINTDEN DROP BACKGROUND PCR Product Concentration (micrograms per milliliter) Total Dilution Initial PCR Product Concentration (micrograms per milliliter) G7+ 6747871 3.747871 6 22.487226 G7- 2783053.333 -0.216946667 6 -1.30168 G71-1 4306884.667 1.306884667 6 7.841308 G71-2 4327330 1.32733 6 7.96398 G71-3 3472404.333 0.472404333 6 2.834426 G72-1 3361343.667 0.361343667 6 2.168062 G72-2 3031025.667 0.031025667 6 0.186154 G72-3 3034515.667 0.034515667 6 0.201094