BME100 s2018:Group7 W1030 L5

BME 100 Spring 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: Dragos Baron Name: Lauren Lossner Name: Mery Montenegro Name: Zach Vance LAB 5 WRITE-UP PCR Reaction Report The pre-lab reading helped our group tremendously with learning how to pipette samples. Some of the group members did not originally know how to us a pipette, but after reading about it, we were successfully able to pipette the samples to set up the reaction. It is important for all of us to understand how to use a micropipettor correctly because it can break easily. The first stop on the micropipettor is passed and used in order to collect the sample solution. The second stop is pressed down in order to add an extra puff of air in order to get all of the sample solution out of the tip of the micropipette. As we found out through human error, if the second stop is pushed down before the sample solution is collected, there would be a higher volume in the micropipettor than it is set to, causing error in calculations and measurements. The final did not have an exact, perfect amount of the amount of liquid, but there was only a very small variation, most likely due to human error. There was a small amount of the PCR reaction mix that was left at the bottom of the tubes. Our labeling scheme remained consistent with our experiments. Fluorimeter Procedure Imaging set-up SmartPhone 1: iPhone 7 To start the imaging setup, the phone should be set on camera mode with a timer on. This experiment in particular used an iPhone 7. The phone is then placed on a stand so that the height of the camera matches that of the glass slide. The phone is then adjusted making sure that it is right on the edge of the slide, capturing the sample from a side view. The phone should be around 4 cm away from the sample. Once the experiment is setup, everything should be covered with a lightbox leaving only one flap open. The phone is then checked one more time to make sure it is focused on the sample. Once everything is in focus, the timer is started and the flap is closed so that the picture can be taken in the dark. The same process is then repeated for the other samples with three pictures taken for each. Placing Samples onto the Fluorimeter Place a slide into the flourimeter with the smooth side down. Take out a smartphone with a good camera. Adjust the camera and flourimeter so that they are at the same level. Make sure that the camera is as close to 90° as possible. Position the camera as close to the flourimeter without it being too blurry and record the distance in cm. Make sure that the flash on the camera is turned off. Put gloves on, and using the micropipette place 160 µL of water in between the first two rows. Turn on the blue LED light. The switch is found on the side of the flourimeter. Using the micropipette, place 80 µL of SYBR GREEN I in between the first two rows. Using the micropipette, add 80 µL of the desired calf thymus solution. Position the slide so that the blue LED light is being focused on the drop. Place the box over the flourimeter and phone and set your phone to a 3-10 second delay when taking the picture. After you start taking the picture, quickly let down the flap of the box so that no light gets in. Repeat this step two more times for a total of three pictures. Remove the box so that you have access to the flourimeter and your phone. Using the micropipette, remove the 160 µL drop and move the slide to the next available position. Repeat steps 6-13 for all of the calf thymus DNA solutions. Get the 8 PCR reaction samples and label each tube with a red dot to match with the PCR Reaction Sample labels. Set the micropipette to 120 µL and attach a disposable tip. Transfer 100 µL from the PCR tube into the buffer tube of the same name. The buffer tubes are the ones marked by a red dot. Repeat this for all of them, discarding tips after every transfer. It is important that there is no cross contamination. Repeat the same procedure used earlier for the calf thymus DNA solutions to all 8 of the PCR tubes, Data Collection and Analysis Images of High, Low, and Zero Calf Thymus DNA High Calf Thymus DNA Low Calf Thymsus DNA Zero Calf Thymus DNA Due to human error, there was no zero calf thymus data collected. A sample was taken from a different group but the pictures were different and the data would have skewed our data. This error affected all of our results. Calibrator Mean Values Sample Number RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation Image 1 Image 2 Image 3 C-1 6881295 6594279 7018360 6831311.333 176701.158 C-2 5429623 5960987 5829648 5740086 225983.6853 C-3 4544709 4753178 5571527 4956471.333 443159.0654 C-4 2987156 3219003 3092902 3099687 94772.65787 C-5 2388863 2291859 2051810 2244177.333 141671.7737 Calibration curves Images of Our PCR Negative and Positive Controls Positive Control Negative 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) G 1-1 4059600.00 1.0298 12 12.3576 G 1-2 3193538.67 0.596769333 12 7.161232 G 1-3 3465345.67 0.732672833 12 8.792074 G 2-1 2195238.00 0.097619 12 1.171428 G 2-2 1785487.00 -0.1072565 12 -1.287078 G 2-3 3024767.33 0.512383667 12 6.148604 Positive 5320315.00 1.6601575 12 19.92189 Negative 2524020.33 0.262010167 12 3.144122 PCR Results: Summary Our positive control PCR result was 19.92 μg/mL Our negative control PCR result was 3.14 μg/mL Observed results Patient 46793 : The image of the drop as seen below shows some green coloring. The results look similar to that of the positive control but the quantitative results tell a different stories. The quantitative results are in between the results gathered in the positive and negative controls trials. Because of this, the test proved to be inconclusive. Patient 31082 : The image of this patient's drop as seen below shows no green coloring. The results look very similar to that of the negative control but the quantitative results tell a different stories. The quantitative results correlated with that of the negative results. Because of this correlation, a conclusion was made that patient 31082 is positive. Conclusions Patient 46793 : Inconclusive Patient 31082 : Negative References