BME100 f2015:Group6 1030amL5

BME 100 Fall 2015	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 Caroline Dolt(s) Kamrie Ickes(s) Rex Moore Connor Beck Name: Chricitan B. Adane Andres X. Gandara LAB 5 WRITE-UP PCR Reaction Report Our team worked sufficiently to set up the reaction using pipetting techniques. We had 8 tubes, one being the positive control and another being the negative control. The other six tubes contained the 100 nanoliter PCR reactions. After each reaction was set up and the lids on each tube were closed tightly, we transferred them to the PCR machine. The pre-lab was beneficial because it prepared us for the use of a pipettor by listing each each step required to get successful measurements. We understood that the first stop of the pipettor should be used for obtaining the volume of solution noted on the side of the device, while the second stop is used for injecting the solution into the tubes. It is because of the accuracy of the pipettor the the final reactions had the same amount of liquid. We did not see any excess liquid in the tubes after transferring it. Labeling was also successful, as we followed the instructions given to us. Fluorimeter Procedure Smart Phone Camera Settings Type of Smartphone: iPhone 5 Flash: Off ISO setting: No setup option White Balance: No setup option Exposure: No setup option Saturation: No setup option Contrast: No setup option Camera set-up The phone was placed in the cradle and oriented to face the fluorimeter. The camera on the phone was higher than the fluorimeter so two small tupperware tops and a notebook were placed under the fluorimeter to make the camera level with the fluorimeter. A usb drive was also placed in the cradle along with the phone so the phone would not be at an angle when taking pictures of the drop. The usb drive was between the back of the phone and the cradle so the drop on the fluorimeter and the camera were on the same horizontal plane. Distance between the smart phone cradle and drop = 4 cm Placing Samples onto the Fluorimeter A slide was positioned on the fluorimeter 80 microliters of Sybr Green were added between two of the rows on the slide 80 microliters of the DNA sample were added A three second timer was set on the phone and a picture was taken Two more pictures were taken for each DNA sample The 160 microliters of Sybr Green and DNA sample were removed from the test tube using the micropipette The slide was moved so the next two rows were in line with the light Steps 2-7 were repeated until all DNA samples had been photographed three times 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 RAWINTDEN DROP - BACKGROUND RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation Image 1 Image 2 Image 3 5 2.5 C-1 7695436 8778810 8648377 8374207.66666667 591440.126702892 2 1 C-2 9976618 10139271 10217875 10111254.6666667 123044.387163061 1 0.5 C-3 5960300 5976139 6315199 6083879.33333333 200485.185538317 0.5 0.25 C-4 5413972 5865862 5191720 5490518 343527.766895196 0.25 0.125 C-5 4962662 5351433 5223847 5179314 198174.452634541 0 0 C-6 2767243 3014607 82822 1954890.66666667 1625969.88184294 Calibration curves Images of Our PCR Negative and Positive Controls (1) G6- Control (2) G6+ Control PCR Results: PCR concentrations solved PCR Product TUBE LABEL MEAN (of RAWINTDEN DROP _ BACKGROUND PCR Product Concentration (µg /mL) (Step 5 calculation) "Total Dilution "Initial PCR Product Concentration (µg /mL) (Step 6 calculation) (Step 6 calculation)" G6+ 6588265 1.588265 12 19.05918 G6- 5219016.6667 0.21901667 12 2.6282 G6 1-1 6623092 1.623092 12 19.477104 G6 1-2 3326347.333 1.673652667 12 20.083832004 G6 1-3 3174961.333 1.825038667 12 21.900464004 G6 2-1 9316279 4.316279 12 51.795348 G6 2-2 9177447 4.177447 12 50.129364 G6 2-3 8846335.6667 3.8463356667 12 46.1560280004 PCR Results: Summary Our positive control PCR result was 19.05918 μg/mL Our negative control PCR result was 2.6282 μg/mL Observed results Patient 41418 : The photos for this patient show green drops. The average concentration was 20.48713334 μg/mL. Patient 39567: The photos for this patient show clear drops. The average concentration was 49.36024667 μg/mL. Conclusions Patient 41418 : This patient matches the positive photo. The average of this patient is very close to the positive control, supporting that the SNP is present in this patient's DNA. Patient 39567 : This patient matches the negative photo. However, the concentration of this patient is closer to the positive control concentration than the negative control concentration. Visually, the patient is negative but quantitatively, the patient is positive. These results are inconclusive and a retest would be advised.