BME100 s2015:Group10 12pmL5

BME 100 Spring 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 Error creating thumbnail: File missing Name: Joseline Valenzuela Error creating thumbnail: File missing Name: Itai Kreisler Error creating thumbnail: File missing Name: Clayton Nunn Error creating thumbnail: File missing Name: Isaac Clouse LAB 5 WRITE-UP Procedure Smart Phone Camera Settings Type of Smartphone: Galaxy S5 Flash: Off ISO setting: 800 White Balance: Auto Exposure: +2.0 Saturation: Auto Contrast: Auto Calibration Turn on the Blue LED light on the fluorimeter. Adjust the camera settings on the smartphone if possible. Turn on camera. Place the smartphone on the cradle and adjust its distance from the fluorimeter and record it. Use plastic trays to adjust the height of the fluorimeter so the smartphone take a picture of the side of the slide. Be careful to keep the position of the smartphone consistent, otherwise the results could be affected. Distance between the smart phone cradle and drop = 6 cm Solutions Used for Calibration Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Volume of the 2X DNA solution (µL) Volume of the SYBR GREEN I Dye solution (µL) Final DNA concentration in SYBR Green I solution (µg/mL) 5 80* 80* 2.5 2 80* 80* 1 1 80* 80* 0.5 0.5 80* 80* 0.25 0.25 80* 80* 0.125 0 80* 80* 0 'Placing Samples onto the Fluorimeter Use a micro pipettor to transfer 80 microliters of SYBR Green onto the rough side of the slide. Make sure to apply the solution between two circles in order to form a spherical shape. Replace micropipettor tip. Add 80 microliters of one of the calf thymus solutions to the SYBR green drop. Move the slide so the solution is aligned with the blue LED. Take a picture by using the timer tool on the camera. Cover the fluorimeter from any light while taking the picture by placing a lid on the box. Take 3 pictures of every sample of the drop while the camera is focused. Once finished taking the picture, carefully remove the lid on the box. Try not to change the position of the smartphone. Remove the 160 microliters of solution using a micropipettor. Replace micropipettor tip. Repeat steps 7-14 for all of the concentrations of calf thymus DNA. Data Analysis Representative Images of Negative and Positive Samples Negative Sample: Positive Sample: Image J Values for All Calibrator Samples Final DNA concentration in SYBR Green I solution (ug/mL) AREA Mean Pixel Value Rawintden of the drop Rawdinten of the background Rawdinten drop - background 5 134997 84.524 11454707 153690 11301017 5 136212 86.704 11810103 158792 11651311 5 136613 85.68 11705069 135600 11569469 1 154664 61.984 9586727 190422 9396305 1 163276 65.584 10708223 210737 10497486 1 162561 64.813 10536084 234323 10301761 0.5 159526 40.806 6509655 179213 6330442 0.5 154390 42.281 6527715 189658 6338057 0.5 147508 42.307 6240665 198054 6042611 0.25 132456 30.609 4054351 155804 3898547 0.25 133620 29.464 3936991 123573 3813418 0.25 132628 31.968 4239909 140205 4099704 2 151695 89.107 13517048 217071 13299977 2 149465 91.944 13742408 244300 13498108 2 154378 91.811 14173670 232948 13940722 0 125700 12.048 1514380 142304 1372076 0 140865 11.92 1679060 165324 1513736 0 144596 12.457 1801259 158680 1642579 Calibration curve ***Note: Our camera was not sensitive enough to capture the high concentrations of SYBR green. As a result we elected to exclude the last data point in order to obtain a more accurate trend line.*** PCR Results Summary PCR Product TUBE LABEL RAWINTDEN DROP- BACKGROUND PCR Product Concentration (ug/mL) Initial PCR Product Concentration (ug/mL) 10+ 18002353 2.500392167 1.5002353 10 1-1 18046396 2.507732667 1.5046396 10 1-2 19446937 2.741156167 1.6446937 10 1-3 20715534 2.952589 1.7715534 10 - 10348475 1.224745833 0.7348475 10 2-1 9226110 1.037685 0.622611 10 2-2 7733764 0.788960667 0.4733764 10 2-3 9526790 1.087798333 0.652679 Our positive control PCR result was 1.500 μg/mL Our negative control PCR result was .7348 μg/mL Observed results Patient 67713 : This patient's samples were fluorescent (green). The average initial PCR product concentration was 1.64(μg/mL), which is above the initial PCR concentration of the positive control of 1.500 (μg/mL), like all samples of this patient. Patient 89702 :The images for patient two looks darker than our negative control for these samples. Also, our values for the μg/mL were .62, .47, .65 for each sample of this patient. Since all the values were under our negative control, it is correct to assume that this patient did not suffer from the SNP. Conclusions Patient 67713 : Based on the similarities of the initial PCR concentrations of the positive control and patient 67713, it is safe to assume that the patient tested positive for SNP. As mentioned above, the high fluorescence and the values of PCR concentration of the positive control closely matched this patient. Patient 89702 :As mentioned above, each concentration calculated from the calibration curve yielded a concentration lower than that of our negative control. Thus we ascertained that this patient did not have the SNP, based on the low numbers in the previous section. SNP Information & Primer Design Background: About the Disease SNP SNP stands for "single nucleotide polymorphism". This occurs when there is a discontinuous genetic variation on a single nucleotide, resulting in different types of individuals. This variation is found in Homo sapiens on chromosome 8. Variations in SNP can cause Hyperlipidemia. This SNP is associated with genes LIPD and HDLCQ11. According to summaries of research on PubMed, SNP is linked to coronary heart disease. It is believed to affect cholesterol levels, which in turn can lead to stroke and other related illnesses. Primer Design and Testing