BME100 f2018:Group8 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: Joseph Simpson Roles: Writer/Researcher Name: Rachel Miller Roles: Writer/Researcher Name: Karla Cosio Roles: Writer/Researcher Name: Adriana Eseberre-Arellano Roles: Writer/Researcher Name: Katya Martinez Roles: Writer/Researcher Name: Griffin Sells Roles: Writer/Researcher LAB 5 WRITE-UP PCR Reaction Report It was the team’s first time pipetting, thus, the pre-lab reading was very educational because the micropipetting videos showed how to use proper micropipetting technique to avoid mistakes and introduced the uses for the first and second stop of the pipette. It was decided that Katya and Karla were going to do the pipetting for the team. Thus, they gathered the materials which included the 8 tubes of PCR reaction mix, each with 50 μL of Taq DNA polymerase, MgCl2, and dNTP. Then, another 8 tubes had the DNA/primer mix, each with 50 μL of different templates of DNA but the same forward and reverse primers. Moreover, the 8 empty tubes in a rack. Lastly, the micropipette, box of fresh pipette tips, and the waste container. Karla began labeling the 8 empty tubes first starting with “G8 P”, followed by “G8 N”, “G8 1-1”, “G8 1-2”, “G8 1-3”, “G8 2-1”, “G8 2-2”, and “G8 2-3” for the last tube. As a result, we did not have to change our labeling scheme. Katya did the first half of pipetting, transferring the PCR reaction mix to the empty tube, then discarding the pipette tip. Then, taking the DNA/primer mix and transferring it in the correct labeled tube. The only difficulty at first was making sure all the liquid was obtained. Though, the other parts of pipetting such as going to the first stop, second stop and discarding the tips replaced with new ones was successful. Karla, then, did the last half of the tubes, making sure that each solution went to the correct tube, avoiding cross contamination. Likewise, she was successful of knowing when to use first and second stop, but it was just a bit difficult obtaining all the liquid from the tubes. When the tubes containing the DNA samples and PCR reaction mix were compared to each other, all had the same amount of liquid. In the other tubes, they were empty of any liquid that they had initially. Next, it was made sure that all the tubes were completely closed, ready for the next step. Dr. Garcia announced once groups were done pipetting, they were to bring their 8 tubes of solution to a PCR machine. He explained there that groups would have to share one machine, thus, our team was paired with another group. We placed our 8 tubes in the machine arranged in the first two rows (four per row). Then, the other group did the same in the last two rows. Once the top was shut, it was helpful that Dr. Haynes had provided instructions in the workbook on which to set the settings of the PCR machine. From there, we just waited one hour until the PCR machine was complete. Fluorimeter Procedure Imaging set-up Smart phone model: iPhone 7 Before placing the phone on the cradle, the settings on the camera had to be adjusted. Thus, both the exposure and saturation were set to the highest setting. Moreover, it was made sure that the contrast was set to the lowest setting and white balance set to auto. The timer was set to 3 seconds. Lastly, the most critical thing was to make sure the flash was turned off. After the settings on the camera were all set, the phone was placed vertically on the cradle and adjusted so that the the view on the phone is directly capturing the drop placed on the fluorimeter. It was adjusted by placing two trays under the fluorimeter to be in level with the camera. In addition, the phone was in level to capture the side of the drop and pressed to focus on the drop. It was made sure that when focusing, the phone was measured exactly 4.6 cm away from the fluorimeter to get a focused and close image of the drop. Placing Samples onto the Fluorimeter Find the center of the first two rows of the slide. Place an 80 microliter drop of SYBR GREEN 1 with the pipettor onto the center of first two rows of the slide. Look to see that the drop has formed into the shape of a ball and is placed in the correct position. Add an 80 microliter drop of one of the calf thymus (or water blank) solutions to the SYBR GREEN 1 drop that is on the slide. Look to see that now one large drop (sample) is centered in between the first two rows. Position the drop so that the blue light of the fluorimeter is illuminating directly through the center of the drop to the other side. Place the cradle with the phone 4.6 cm away from fluorimeter making sure it is focused on drop. Take the light box to cover the phone and fluorimeter but leaving one side exposed to light. Press the timer on phone when drop is focused and close the flap of the light box so that no light enters. Save the image that is taken from the phone. Reset timer on phone and repeat steps 9 and 10 two more times. Check for a total of three images for that specific drop. Remove the 160 microliter drop with a fresh pipetter tip on the pipettor from the slide. Discard waste into waste container. Place a fresh pipette tip on pipettor. Move the slide to the next unused rows. Once there are no unused rows, get a new slide to insert into the fluorimeter. Repeat steps 1-16 for all other samples (calf thymus DNA and PCR reaction). Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Picture of 5 μg/mL sample. Picture of .5μg/mL sample. Picture of 0μg/mL sample. Calibrator Mean Values Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation Image 1 Image 2 Image 3 5 2.5 C-1 17534074 19253884 20929515 19239157.67 1697768.401 2 1 C-2 22844028 21522363 21359872 21908754.33 814035.291 1 0.5 C-3 19152223 18769882 18525896 18816000.33 315700.1001 0.5 0.25 C-4 15172998 15378714 15531823 15361178.33 180054.0748 0.25 0.125 C-5 16001257 15881368 16894558 16259061 553611.48 0 0 C-6 11718086 11485830 11327953 11510623 196244.6408 Calibration curves Data Plot 1 for Calibration Curve. Data Plot 2 for Calibration Curve. Images of Our PCR Negative and Positive Controls Picture of Negative PCR sample. Picture of Positive PCR sample. PCR Results: PCR concentrations solved PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP - BACKGROUND) PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration (µg /mL) G8 P 18381238 1.6762476 12 20.1149712 G8 N 13061566 0.6123132 12 7.3477584 G8 1-1 10740676 0.1481352 12 1.7776224 G8 1-2 11602016 0.3204032 12 3.8448384 G8 1-3 8172676 -0.3654648 12 -4.3855776 G8 2-1 17759392 1.5518784 12 18.6225408 G8 2-2 15954917 1.1909834 12 14.2918008 G8 2-3 11514181 0.3028362 12 3.6340344 PCR Results: Summary Our positive control PCR result was 20.11 μg/mL Our negative control PCR result was 7.35 μg/mL Observed results Patient 39480 (1): For patient 39480 the average of the three diluted PCR product concentrations was .03436 μg/mL. This means that the average initial PCR product concentration was 1.23688 μg/mL. These images show that the sample is positive because when the control image of the positive and this image are compared the sizing of both drops this image can be shown to be very similar. Patient 81770 (2): Patient 81770 had an average diluted PCR value of 1.01523 μg/mL. Taking this number and the amount of dilutions into account the average initial PCR product concentration was 12.18279. This is further proven when the image is analyzed using pixel measurements. These images show that the sample is negative because when the control image of the negative and this image are compared the sizing of both drops this image can be shown to be very similar. This is further proven when the image is analyzed using pixel measurements. Conclusions Patient 39480 : This patient's value of 1.23688 puts it closer to the negative control than the positive control this leads our group to believe that this patient was negative. Patient 81770 : Patient 81770 has a value of 12.18279 which means that its value lies between the positive and negative controls. This fact leads to inconclusive test results for this patient.