The pre-lab reading and activities proved to be very helpful. The majority of the members of our group had little to no experience using a micropipette prior to this lab. However, after reviewing the pre-lab materials everyone felt comfortable using a micropipette. The difference between the first and second stop on the pipettor was easy to understand. In order to suck the liquid up into the pipettor, the button was pushed to the first stop. The second stop was pushed to release the contents of the micropipette into a second vessel. To the naked eye, the final reactions all appeared to have the same amount of liquid. (All the PCR tubes appeared to have the same amount of liquid in them.) The experimenters made sure that the pipettor was calibrated to the correct volume as well as making sure that all the liquid from the pipettor was released into the container. All the contents from the and PCR reaction mix were used. The labeling scheme did not change. The labeling scheme did not change. Each empty tube was labeled with the patient ID or positive/negative control and a number (1,2 or 3) to specify the replicates of the patient ID reactions.
Fluorimeter Procedure
Imaging set-up
A smart phone was placed in a cradle. The back of the phone was 6 centimeters away from the drop itself. The fluorometer was placed on two plastic trays so that the camera was level with the drop. This way, the camera was able to take pictures of the drop from the side. The camera’s flash was turned off. The whole apparatus was placed in a dark box.
Placing Samples onto the Fluorimeter
The slide was placed into the fluorimeter with the smooth side of the slide face down.
80 microliters of SYBR green was placed in between the first and second circles on the slide. 9.The fluorimeter light was turned on. The light went through the drop, if the light did not go through the drop, subsequent adjustments were made.
80 microliters of calibration solution were placed on top of the 80 microliters of SYBR green already on the side.
The fluorometer was then covered by the lightbox.
The images taken with the camera timer were blurry. In order to increase the quality of the pictures, the fluorometer and smartphone were pushed the end farthest from the opening of the lightbox. By doing this, the fluorometer was in the darkest place in the box.
A group member would position the smartphone in the cradle 6 centimeters away from drop and took 3 pictures of the drop.
After the picture was taken, the drop was removed from the slide.
The slide was then moved to the next position: the second and third row of circles.
This process was repeated. However, for each trial 80 microliters were taken from one of the tubes labeled either negative, positive, patient ID: 41134 (1,2, and 3) or patient ID: 45645 (1,2, and 3.). The 80 microliters from one of these labeled tubes from the previous lab was placed on the 80 microliter SYBR green drop on the slide. Three individual pictures were taken of each drop.
Our negative control PCR result was -68.94689 μg/mL
Observed results
Patient 41134: Images had a bright green, fluorescent glow and an average initial PCR product concentration of 18.44736 μg/mL
Patient 45645: Images did not have the green glow and had an average initial PCR product concentration of -69.26603 μg/mL
Conclusions
Patient 41134 : concluded to be positive because the average initial PCR product concentration of the patient is 18.44736 μg/mL which is closer to the positive control value of 4.63764 μg/mL than the negative control value of -68.94689 μg/mL. Because the patient’s results are close to that of the positive control, the patient is concluded to be positive for the presence of the DNA.
Patient 45645 : concluded to be negative because the average initial PCR product concentration of the patient was -69.26603 μg/mL which is closer to the negative control value of -68.94689 μg/mL than the positive control value of 4.63764 μg/mL. Because the patient’s results were close to that of the negative control, the patient is concluded to be negative for the presence of the DNA.
BME 100 Fall 2016
