BME100 f2018:Group13 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: Derek Busch Role(s): Editor Name: Gabriel Baca Role(s): Materials Name: Mia Geric Role(s): PCR Reaction Report Name: Michelle Tieu Role(s): Fluorimeter Procedure Name: Damian Gordillo-Velazquez Role(s): Data Collection & Analysis LAB 5 WRITE-UP Materials Lab Coat Disposable Gloves PCR Reaction Mix, 8 tubes, 50 μl each:Mix contains Taq DNA polymerase, MgCl_2, and dNTP's (hp://www.promega.com/resources/protocols/product‐informaon‐sheets/g/gotaq‐colorless ‐master‐mix‐m714‐protocol/) DNA/ primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer A strip of empty PCR tubes Disposable pipette tips: only use each only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated. Cup for discarded tips Micropipettor OpenPCR Machine: shared by two groups PCR Reaction Report Our team had an easy time pipetting the samples to set up the reactions. The pre-lab reading was very helpful in understanding the procedures we performed today, as it showed visually and audibly how to pipette solutions. By knowing how the lab was going to function, we had an advantage in the execution and understanding of the procedure. In our group, Mia and Michelle pipetted the solutions for Lab C. For Lab D, Gabe and Damian pipetted the solutions. We were able to easily understand the difference between the first and second stops on the pipettor. The final samples all contained the same amount of liquid. There was not any liquid left in the tubes that the DNA samples and PCR reactions were mixed in. We did not change our labeling scheme for our test tubes. Overall, the pipetting was relatively simple and easy, because the group was knowledgeable on the proper technique from the pre-lab reading and quiz. Fluorimeter Procedure Imaging set-up To capture images from the flourimeter, a smartphone with decent camera quality needs to be placed in the phone cradle directly opposite from the light beam from the light box. If possible, the flash should be inactivated, the ISO should be set to above 800, the white balance should be set to auto, the exposure and saturation should be set to the highest setting, and the contrast should be set to the lowest setting. Using the micropipettor, place a 160mL drop of water in between the first two rows of your slide. Switch the excitation light on for the blue LED. Turn on your cell phone camera, turn off the flash, set ISO to 800 or higher, set white balance to auto, change exposure to the highest setting, set saturation to the highest setting, and finally change contrast to the lowest setting. Set your smartphone in the cradle with the camera facing the slide. Adjust the fluorimeter using the plastic trays so that the camera takes a picture of the drop sideways. Adjust the smartphone so that it is as close as possible to the drop, while still remaining in focus. Record the distance between the smartphone and the drop using a ruler. Placing Samples onto the Fluorimeter Using the micropipettor, place an 80 microliter drop of SYBR Green I in the middle of the first two rows of the slide. Add 80 microliters of calf thymus solution to the drop to form a sample. Align the drop so the blue led is focused. Set a timer on your camera to take a picture after the light box is lowered. Take three pictures of the drop. Remove the box, repeating the steps for the rest of the calf thymus DNA. Be sure to take three independent pictures for each sample for accuracy. Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA 5 μg/mL sample 0.5 μg/mL sample zero DNA Calibrator Mean Values Sample Number Image number Final DNA concentration in SYBR Green I solution (µg/mL) AREA Mean Pixel Value RAWINTDEN OF THE DROP RAW INTDEN OF THE BACKGROUND RAW INTDEN OF DROP - BACKGROUND C-1 1 2.5 15619 108.259 2182112 16852 2165260 C-1 2 2.5 15619 106.871 1669212 26755 1642457 C-1 3 2.5 15619 107.15 1673578 29364 1644214 C-2 1 1 13000 96.532 1660418 15706 1644712 C-2 2 1 13000 98.662 1282601 24797 1257804 C-2 3 1 13000 99.286 1290719 35267 1255452 C-3 1 0.5 13016 67.088 1091093 13081 1078012 C-3 2 0.5 13016 65.979 858787 17376 841411 C-3 3 0.5 13016 65.997 859014 22674 836340 C-4 1 0.25 17248 143.822 2480636 31760 2448876 C-4 2 0.25 17248 143.955 2482937 54794 2428143 C-4 3 0.25 17248 143.871 2481487 36320 2445167 C-5 1 0.125 24341 130.123 3167314 34281 3133033 C-5 2 0.125 24341 129.906 3162054 36420 3125634 C-5 3 0.125 24341 130.106 3166904 40053 3126851 C-6 1 0 12984 75.043 974360 24886 949474 C-6 2 0 12984 71.294 925681 22969 902712 C-6 3 0 12984 74.652 969284 21450 947834 Calibration curves Images of Our PCR Negative and Positive Controls Negative Control Positive Control PCR Results: PCR concentrations solved PCR Product TUBE LABEL Image Number AREA Mean Pixel Value RAWINTDEN OF THE DROP RAWINTDEN OF THE BACKGROUND RAWINTDEN DROP - BACKGROUND G13 + 1 21980 93.822 2062211 42276 2019935 G13 + 2 21980 93.439 2053781 47206 2006575 G13 + 3 21980 93.815 2062064 46058 2016006 G13 1-1 1 25026 90.712 2270164 36160 2234004 G13 1-1 2 25026 89.746 2245986 47712 2198274 G13 1-1 3 25026 90.557 2266277 25327 2240950 G13 1-2 1 24263 97.112 2356237 32919 2323318 G13 1-2 2 24263 104.251 2529441 34205 2495236 G13 1-2 3 24263 97.604 2368175 32392 2335783 G13 1-3 1 24056 103.722 2495126 61195 2433931 G13 1-3 2 24056 105.78 2544645 47381 2497264 G13 1-3 3 24056 104.361 2510498 52545 2457953 G13 - 1 23589 116.75 2754018 42957 2711061 G13 - 2 23589 116.88 2757085 35657 2721428 G13 - 3 23589 116.164 2740187 41418 2698769 G13 2-1 1 24360 105.613 2572724 43329 2529395 G13 2-1 2 24360 106.302 2589512 53160 2536352 G13 2-1 3 24360 106.037 2583056 47936 2535120 G13 2-2 1 25806 105.317 2717815 30529 2687286 G13 2-2 2 25806 104.064 2685476 39027 2646449 G13 2-2 3 25806 106.034 2736318 39423 2696895 G13 2-3 1 26668 103.397 2757379 39076 2718303 G13 2-3 2 26668 104.271 2780708 36150 2744558 G13 2-3 3 26668 103.944 2771981 32095 2739886 PCR Results: Summary Our positive control PCR result was 27.47881297 μg/mL Our negative control PCR result was 46.3435127 μg/mL Observed results Patient #1 81477 : The image for patient 1 shows a greener tint around the bubble that observed. For patient 1 the PCR product concentration was 27.47881297 μg/mL. Patient #2 68313 : The image observed for patient 2 shows that it doesn't have a strong tint, it is mostly clear. For patient 2 the PCR product concentration was 46.3435127 μg/mL. Conclusions Patient #1 81477 : Patient #1's PCR Product Concentration values were most similar to the positive control value. The patient is most likely positive. Patient #2 68313 : Patient #2's PCR Product Concentration values were most similar to the negative control value. The patient is most likely negative.