BME100 f2017:Group11 W1030 L5

BME 100 Fall 2017	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 Error creating thumbnail: File missing Name: Teleah Hancer Error creating thumbnail: File missing Name: Jose vallejo Error creating thumbnail: File missing Name: Una Error creating thumbnail: File missing Name: Zoe Marmitt Name: student Name: student LAB 5 WRITE-UP PCR Reaction Report Going through the pre lab material was not only extremely important to understand how to pipette, but it made the lab run smoothly. Doing the PCR virtual lab helped explain the reason why we are pipetting. The virtual micro-pipetting exercise familiarized us with the micro pipettor.We all practiced using the pipette with no liquid to understand the difference between the first and second stop. Practicing micro-pipetting allowed us to be more precise when mixing the PCR primer mixture and the DNA, and ensured that all the final reactions had the same amount of liquid. To double check that the amounts were the same, we checked to make sure there was no liquid left in the tubes that the DNA samples and PCR reaction mix was taken from. Since everything was properly labeled at the start of the lab, we did not have to change our labeling scheme. Fluorimeter Procedure Imaging set-up It was important to put on all PPE before starting. Then all the materials were gathered. This included the fluorimeter, 3 glass slides, an iPhone 6, 3 small pipette holder boxes. First, one side of the fluorimeter box was opened and the lid was removed so everything could be easily set up. Then the smooth side of the slide was put face down in the fluorimeter. Next, the phone camera was turned on and set to a 3 second timer. It was then placed on the phone holder and an 80 microliters sample of H2O was pipetted on the first 2 clear circles in the middle of the slide. Then 3 small pipette holder boxes were placed beneath the fluorimeter to get the droplet level with the camera, and the camera distance was adjusted so a clear image of the droplet side could be taken. Once the camera was clearly focused on the H2O sample, the H2O was removed and the fluorimeter was ready for imaging. The lid of the fluorimeter was put on every time a picture was taken to prevent any source of light getting in. Placing Samples onto the Fluorimeter Set the micro pipette to 80 micro liters Put on a clean pipette tip Push plunger button to first stop Insert tip into SYBR Green I solution Slowly release plunger button until all 80 micro liters are inside the pipette Place all 80 micro liters of SYBR Green I solution on first 2 clear circles in the middle of the slide by pushing plunger button all the way down to the second stopper Eject pipette tip into discard cup Put on new clean pipette tip Push plunger button to first stop Insert into sample/calibration solution Slowly release plunger button until all 80 micro liters are inside the pipette Place all 80 micro liters of sample/calibration solution over first drop After the pictures have been taken, suck up the whole drop on the slide using pipette Eject pipette tip into discard cup Adjust slide to the center of the next two holes Change slide after all five possible measurement positions have been used Repeat steps 2-16 until all calibration solutions have been put on the fluorimeter Repeat steps 2-16 until all DNA sample solutions have been put on the fluorimeter Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA Error creating thumbnail: File missing 5 μg/mL sample Error creating thumbnail: File missing 0.5 μg/mL sample Error creating thumbnail: File missing 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 Image 1 RAWINTDEN DROP - BACKGROUND Image 2 RAWINTDEN DROP - BACKGROUND Image 3 MEAN Standard Deviation 5 2.5 C-1 17926243 16631330 16348318 16968630 841302.8364 2 1 C-2 14445901 13255933 13720561 13807465 599725.0947 1 0.5 C-3 12373343 9617279 10469292 10819971 1411100.347 0.5 0.25 C-4 10345440 12265350 12057955 11556248 1053705.74 0.25 0.125 C-5 7173099 6753052 7109988 7012046 226504.5212 0 0 C-6 0 0 0 0 0 Calibration curves Error creating thumbnail: File missing Calf Thymus DNA Calibration Curve 1 Calf Thymus DNA Calibration Curve 2 Images of Our PCR Negative and Positive Controls PCR Results: PCR concentrations solved Negative Control Positive Control Error creating thumbnail: File missing PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP-BACKGROUND) PCR Product Concentration (µg /mL) Total Dilution Initial PCR Product Concentration (µg /mL) G11 1-1 6991641.667 0.49582 12 4.77994 G11 1-2 28544989 11.272495 12 135.2699 G11 1-3 7234025 0.6170125 12 7.404 G11 2-1 4515353 -0.7423235 12 -8.90788 G11 2-2 7005001 o.5025005 12 6.030006 G11 2-3 7731292.33 0.8656165 12 10.3877 G11 + 16592518.33 5.296259165 12 63.555 G11 - 5221622 -0.389189 12 -4.670 PCR Results: Summary Our positive control PCR result was 63.555 μg/mL Our negative control PCR result was -4.670 μg/mL Observed results Patient 43374: The patients results of the PCR Product were all positive and well above O. G11 1-2 however, seems to have way more concentration than the others so that may have been caused because an error in the lab. The pictures of their droplets all seem to relatively clear with little to no white shading in it. Patient 37706:The droplets seem to have a darker shade especially in the lower region of the droplet. The data for this patient only had one concentration that was negative and the rest was positive. All the PCR product concentration are close to zero except for Conclusions Patient 43374 :They tested positive for the disease because the result are very similar to that of the positive. the mean is close to the positive. Patient 37706 :They tested negative for the disease because they had results similar to the negative results