BME100 f2018:Group6 T1030 L5

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OUR TEAM Name:Zachery Shropshire Name: Adriana Gillespie Name: Anna Haworth Name: Sarah Ruby Name: Siris Smith LAB 5 WRITE-UP PCR Reaction Report During the first few beginning attempts, a couple of group members were focusing on pipetting which kept the lab going at a fairly steady pace, however, as time went on, another member had been included in order to create a system that was faster, helping the entire process flow smoother. It began to get a little troublesome however to constantly switch from 80 mL to 160 mL in order to clean up the DNA sample/SYBR Green 1 mix, but added to the experience of being able to use the pipette better. Being provided with the pre-lab reading had greatly assisted in preparing the group for how the lab will be carried out and what to expect. The pre-lab reading had as well offered some more insight into what we were working with/ handling in regards to SYBR Green 1 and why it's a more popular dye to work with when -as stated in the reading- looked at from a "green chemistry" perspective. Proper utilization of the pipette due to experience helped smooth out the experience and allowed more time to comprehend what the interaction of the SYBER Green 1 and DNA samples meant when either the SYBER Green 1 wasn't as fluorescent or had not shown up at all. The group understood that the first stop in the pipettor is used in order to discharge the amount of liquid that it was being held to while the second stop is to be used as an extra force to make sure that all the liquid has been removed. During the pipetting process there were a few times when the second stop was used when making sure either all the SYBER Green 1 was used or the DNA samples. From what could be observed there was very little, if not nothing left in the tubes. During the entire process group members pipetting made sure to keep the pipette at 80mL in order to be sure that the correct amount of each sample and SYBER Green 1 was used; being able to use the pipette's second stop had also ensured the amount of liquid used was all 80 mL. The labeling scheme in regards to taking 3 images of the drops, had "changed," that in order to separate them, an arbitrarily taken image had been placed in-between. Fluorimeter Procedure Imaging set-up The blue LED slide holder was first set down, a slide with a water droplet was placed inside. Using the phone stand and an iPhone, the stand and the slide holder was arranged with the slide holder on top of some plastic boxes so that the water droplet was clearly in view. To get a focused, but close up, image, the base of the stand was set up 10cm away from the slide, and the zoom function on the iPhone's camera was used to get a larger image of the drop so the color could be better analyzed. Then, the black box provided was carefully placed over the setup, and then slid forward so that the back wall of the box just touched the slide holder without pushing it forward--this put the sample as far back in the box as possible, which helped to block out most of the light. The iPhone was set with the recommended settings, with maximum exposure, ISO, and saturation, and minimum contrast. The iPhone used a light for the timer countdown, so the photo was manually taken instead. Each photo was taken with the lid of the black box closed as much as possible. Between trials, the entire box was picked up and placed to the side, while the phone cradle and slide holder were carefully left untouched. For each new trial, once the old drop was cleaned up and the new one properly placed, the black box was placed down exactly as it had been--with the back edge carefully put so it was just touching the slide holder. Placing Samples onto the Fluorimeter Using the pipettor, 160mL of water was placed onto the rough side of the slide while it was in the slide holder so that the drop was pinned in place between two rows of circles. The black box was put on top and closed as much as possible, and 3 photos were taken. It was recorded that water was the first sample, and a photo of the team was taken to separate this data from future data collected. The pipettor tips were discarded after every action to prevent any contamination. The drop was removed using the pipettor, with the waste put in the waste bucket. The pipettor was used to place 80mL of SYBR green in the next row of dots that did not at all touch the dots where the first sample was placed. The slide was shifted first so that the drop could remain in the light of the blue LED. 80mL of the 5 micrograms/mL calf thymus (used for callibration) was to the same drop. It was photographed in the same manner mentioned above, with a photo of the team used as a divider. The steps above were repeated for the rest of the calibration samples as well as the patient samples and positive and negative tubes. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA 0µg/mL sample. 0.5µg/mL sample 5µg/mL Sample Calibrator Mean Values Initial Concentration of 2x Calf thymus DNA(µg/mL) Final DNA Concentration in SYBR Green(µg/mL) Sample # RAWINTDEN Drop-background IMG1 "" IMG2 "" IMG3 Mean Standard Deviation 5 2.5 C-1 7097803 8554814 7282830 7645149 793206.5 2 1 C-2 11129129 1507781 4045301 5560737 4986481 1 0.5 C-3 52905 1795783 - 924344 1232401 0.5 0.25 C-4 2974178 2429918 2200712 2534936 397283 0.25 0.125 C-5 963906 3217284 2934882 963906 1227614 0 0 C-6 1521582 2177726 2243112 1980807 399042 Calibration curves Images of Our PCR Negative and Positive Controls Positive Control Negative Control PCR Results: PCR concentrations solved PCR Product Tube Label Mean (of RAWINTDEN DROP- BACKGROUND) PCR Product Concentration (µ/mL) Total Dilution Initial PCR Product Concentration (µg/mg) positive 3,331,367 0.777 12 9.325468 negative 958,021 -0.014 12 -0.167916 G6 1-1 4936177 1.312 12 15.744708 G6 1-2 4724072 1.241 12 14.896288 G6 1-3 2609195 0.536 12 6.43678 G6 2-1 1498625 0.166 12 1.9945 G6 2-2 2933690 0.645 12 7.73476 G6 2-3 1241861 0.081 12 0.967444 Note: the G6 1 tubes are Patient 1's samples, with the patient ID 23643. The G6 2 tubes are Patient 2's samples, with the patient ID 47311. PCR Results: Summary The positive control PCR result was 9.325 μg/mL The negative control PCR result was -0.1679 μg/mL Observed results Patient 23643: The image came out to look like a greenish fluorescent eclipse or dimensional bubble. Patient 47311: Picture showed a small bubble in the middle with no green fluorescence. Conclusions Patient 23643: Postive. The values were closest to the positive control. Patient 47311: Negative. The values were closest to the Negative control with 1 false positive result.