BME100 f2018:Group11 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: Katelyn Meehan Name:Michail Maltsev Name: Soledad Carrillo Name: Abigail Ouellette Name: Antara Sira LAB 5 WRITE-UP PCR Reaction Report Today’s lab involved using a micropipette to dispense 50 microliters each of PCR reaction mix and the corresponding DNA/primer mix into the eight PCR reaction tubes. As we have five people in our group, all of our members did not need to be involved in the preparation. I, Katelyn Meehan, did the preparation work for the PCR reaction. We decided I should do it because I was the one member who had never before used a micropipette. While completing the lab, I was glad I went through the pre-lab reading because the videos were very detailed and was a great example to go off of. I was able to catch on fast between the first and second stops on the pipettor but I realized it was a slow, long processes to make sure we got every little drop of the mix. As I was using the pipette, I made sure there was no liquid left in the tubes so our final reactions wouldn’t be off with each other. If there was liquid left over, I made sure I went back in with a new tip and got it all out. Overall, the pipetting and labeling scheme worked out smoothly and in the end our final reactions had the same amount of liquid as each other. Fluorimeter Procedure Imaging set-up After many trials, our group managed to set up a phone in order to accurately and clearly take a picture of the drop in the fluorimeter. To begin, we took the best three phones in our group and took a picture of the drop on each of them. In doing so, we were able to determine which phone would be the best one to properly capture the image of the drop. Once we chose the phone we would use for the rest, Samsung S6 Edge, we set up the fluorimeter. We raised the fluorimeter 5.2 centimeters then we set up the phone on the stand 5.5 cm away from the fluorimeter, set the timer changes the settings to take three pictures. Placing Samples onto the Fluorimeter Step one, add two drops of H₂O on the slide that is coated to repel water between the first two rows. Step two, turn the excitation/blue light on. Step three, turn the smartphone camera on, and adjust the settings in order for it to accurately capture an image of the drop. Step four, place the phone in the phone holder at a right angle, 5.2 centimeters away from the fluorimeter. Step five, place an 80 mL drop of SYBR Green in the middle of the slide, then place 80 mL of water on top of that Step six, align drop so the blue LED light is focused in the middle of the drop. Step seven, set the timer on your camera so the picture is taken a few seconds after the fluorimeter. Step eight, take three pictures of each of the drops- make sure the images of the drops are clear and focused. Step nine, remove the box that blocks the light, be careful not to knock the fluorimeter. Step ten, use pipettor to remove 160 mL of the drop. Step eleven, repeat step 5-10 with the other concentrations. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA (1) 5 μg/mL sample (2) 0.5 μg/mL sample (3) Zero DNA 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 454789 455487 457162 455812.6667 1219.559893 2 1 C-2 349466 325339 370942 348582.3333 22814.33874 1 0.5 C-3 329780 372457 418478 373571.6667 44359.50476 0.5 0.25 C-4 206908 240909 223034 223617 17007.99568 0.25 0.125 C-5 245119 232114 209835 229022.6667 17843.97434 0 0 C-6 135685 143112 138249 139015.333363721 3772.337781 Calibration curves Without C-3 Value Images of Our PCR Negative and Positive Controls Negative Control Positive Control 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) G11 P 205704 0.254261489 12 3.051137866 G11 N 42709 -2.61896032 12 -31.42752384 G11 1-1 1675954 26.17134094 12 314.0560912 G11 1-2 143018 -0.850746532 12 -10.20895838 G11 1-3 112040 -1.396816443 12 -16.76179732 G11 2-1 50333 -2.484566976 12 -29.81480372 G11 2-2 62590.67 -2.268492834 12 -27.22191401 G11 2-3 150458 -0.719596679 12 -8.635160147 PCR Results: Summary Our positive control PCR result was .25426 μg/mL Our negative control PCR result was -2.61896 μg/mL Observed results Patient 70758 : The drops of replicated DNA and SYBR Green I stain resembled the negative control. It did not display any green fluorescence to the naked eye or the digital image. The average of the initial PCR product concentrations of the three replicates is 95.6951 µg /mL. Patient 23525 : The drops of patient 23525 DNA with the SYBR Green stain also did not display any apparent green fluorescence. Both to the naked eye and on the photo taken with the mobile phone, the drop did not appear fluorescent. The average initial PCR product concentration of the three replicates was -21.891 µg /mL. Conclusions Patient 70758 : The data value for this patient was much higher than the positive control value. The difference between the two is 95.4409 µg /mL. Theoretically, this should indicate a positive result for patient 70758, that the DNA contained single nucleotide polymorphism. In reality, however, the drops did not indicate any fluorescence, and the data contained significant outliers, skewing the average. As a result, the result is inconclusive. Patient 23525 :The data value for this patient was -21.891 µg /mL quite a bit less than that of the negative control. The difference between the two is 19. 272 µg /mL. This indicates a negative result that is more realistic than the positive counterpart of patient 1. This means that the sample does not contain single nucleotide polymorphism.