The pre-lab reading and quiz was very helpful in preparing our group members for this lab. The practice activities were vital in helping us learn how to use the pipette during the PCR experiment. Only one of our group members worked with the micropipettor while the rest were able to learn through observation. It was very helpful for our group to see examples and instructions before going into the lab.
The first and second stop on the pipettor were easy to understand. The first stop was used to draw up the liquid from the initial test tube. The second stop is then used to release that liquid from the pipettor. By pressing the second stop, a greater volume is released from the pipettor than was entered which allows for all of the liquid to come out. As the lab group went through the steps, we took our time to make sure all the liquid was drawn in and expelled at proper volume to make sure the results would be precise every time. We were also very careful about ejecting the tip from the pipette every time as to not contaminate any of the other substances.
At the end of the reaction, it was fairly clear that each tube did not contain the exact amount of substance every time. We could visually see that some tubes were filled slightly more than other tubes. This probably means that after the PCR reaction mix ran through the thermocycler, there was less than 100 microliters of the reactant. This may have been caused by not being careful enough when drawing the proper amount of liquid during the first lab. These smalls errors could possibly lead to inconsistencies in our data. All of the liquid from each PCR mix tube was extracted completely. We made sure to do this even though not every tube had exactly the same amount of liquid inside.
We did not have to change our labeling scheme. Our labels from the first part of this lab continued to work well.
Fluorimeter Procedure
Imaging set-up
The fluorimeter was turned on and the slide was positioned so that the fluorescent light beams directly on the center of the circles. The camera was then placed exactly 10 centimeters away from the fluorimeter on the phone mount. Then, the phone was set to take 3 photos in 5 second intervals on a timer. The cover of the fluorimeter was then closed and the phone was enclosed in darkness. This allows for the photos to be taken 3 times without the overhead lighting interfering.
Placing Samples onto the Fluorimeter
Place the fluorimeter on the table and turn it on
Place the slide face down in the fluorimeter
Place Iphone 6 on the mount with timer set to take photo
Adjust the fluorimeter until the image looks clear
Use a ruler to measure how far away the Iphone mount is from the fluorimeter. This ensures that it is the exact same distance for all of the photos.
Using the micropipette, take 80 uL of the SYBR Green solution and place it on the center of the 2nd row of circles on the slide
Place 80 uL of the Calf Thymus Liquid on top of the SYBR Green drop
Adjust the slide so the light is centered on the drop
Press the timer button on the smartphone and close the flaps of the box so the slide and phone are completely enclosed
Repeat the step above three times to ensure that you have three images of the drop
Remove the 160 uL drop from the slide and eject the tip from the micropipette and grab a new one
Repeat steps 1-11 for each volume of the Calf Thymus Liquid. When finished, follow these steps again for the PCR reaction mix
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Concentration - 5 μg/mL DNA sample
Low Concentration - 0.5 μg/mL DNA sample
No Concentration - 0.0 μg/mL DNA sample
Calibration curves Images of Our PCR Negative and Positive Controls
Positive:
Negative: Calibration 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
2911339
3014552
2911064
2945652
59669.59742
2
1
C-2
5105538
525290
5348565
2128131
2788987.409
1
0.5
C-3
4688113
4641966
4828659
4719579
97242.82319
0.5
0.25
C-4
3849388
2635361
3842893
3442547
69905.4136
0.25
0.125
C-5
14632474
9968090
14446978
13015847
2641064.324
0
0
C-6
947832
1211383
1205599
1121605
150519.3291
PCR Results: Summary
Our positive control PCR result was 6893300.143 μg/mL
Our negative control PCR result was 763713.6713 μg/mL
Observed results
Patient 76014 (positive control) : no green pigment appeared even though the volume fluctuated from 0μg/mL to 2μg/mL.
Patient 66716 (negative control): no green pigment appeared even though the volume fluctuated from 0μg/mL to 2μg/mL
Conclusions
Patient 76014 (positive control) : Positive; The DNA sequence was very similar to the samples used for this patient.
Patient 66716 (negative control): Positive; The DNA sequence was very similar to the samples used for this patient.
BME 100 Fall 2017
