OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
In this experiment we where attempting to determine the whether the patients we where assigned were either positive or negative for a disease. By utilizing the resources available to us prior to the start of the lab we where able to gain a fairly good understanding of what was expected of us during the lab. While pipetting the Calf Thymus DNA, we figured out almost immediately where the first and second stops where on the pipettor and how to transfer the solutions from one place to another. While using the pipettor we would transfer 80 microliters of SYBR GREEN I to the slide and then add 80 microliters of Calf Thymus DNA to the slide, six different times with 6 different concentrations. This was done to calibrate the fluorimeter in preparation for PCR reaction samples. After conducting the preliminary testing with the Calf Thymus DNA we tested the PCR reaction samples with our patients DNA diluted with a buffer solution. While doing this we had to be sure to label the lids with the patient and trial numbers clearly so we were sure we were testing the correct material. While pipetting 100 microliters of DNA out of the PCR reaction tubes the tubes still contained a small amount of DNA which contained an excess of 100 microliters in it. We then followed the same procedure of putting 80 microliters of SYBR GREEN I solution on the slide and following with 80 microliters of our diluted DNA solution and testing the mixture using the fluorimeter. At the conclusion of our testing, it appeared as though each drop was fairly similar in size. By doing this we where testing to determine the weather or not the patients we where assigned where positive or negative for the disease. In the end we determined that both of our patients where negative.
Fluorimeter Procedure
Smart Phone Camera Settings
- Type of Smartphone: iPhone 6 with an Otter Box case.
- Flash: 3 consecutive flashes counting down the seconds before the picture was taken. (We where unable to remove this feature, since it was an automatic setting)
- ISO setting: iOS 9.1
- White Balance: 0
- Exposure: 0
- Saturation: 0
- Contrast: 0
(All settings that where used where standard for the iPhone 6, nothing besides the timer was manipulated)
Camera set-up
Using the small black camera stand provided, an iPhone 6 was placed in a vertical position, pushed against the back of the stand so that it remains upright. The phone's position was adjusted until the camera of the phone was properly aligned with the edge of the slide in the fluorimeter. Using the phone's timer option, a 3 second timer was used to allow time to close the flap of the fluorimeter cover and prevent light from altering the data.
- Distance between the smart phone cradle and drop = 4.75 cm
Placing Samples onto the Fluorimeter
Lab C
- Set micropipettor to 80 microliters.
- Place glass slide smooth-side down, and rough-side up.
- Using the micropitettor place an 80 microliter drop of SYBR GREEN 1 on the slide between two dots.
- Using a different pipet tip, add 80 microliters of the calf thymus solution to the 80 microliter dot of the SYBR GREEN 1.
- Align the drop with the LED light on the Fluorimeter, making sure that the light is focused on the drop.
- Place the black box over the Fluorimeter and camera set up. Then using the settings on the camera that where previously described focus on the drop and take the picture, being sure to close the flap of the box slowly before the picture is taken to ensure good photo quality. Take three pictures.
- Remove the box, and using the micropipettor remove the drop from the slide and discard it in a waste cup. Remove the slide from the Fluorimeter and discard it in a waste cup.
- Repeat steps 2-6 for the other concentrations of calf thymus solution, being sure to change the tip of the pipet with each new solution.
Lab D
- Set micropipettor to 120 microliters, and place disposable tip on.
- Transfer all 100 microliters of the liquid from each PCR tube to the buffer tubes market with a red dot. Making sure to label the buffer tube with the corresponding DNA and trial numbers. Make sure to change tips between patients and trials.
- Close the caps of the tubes and invert to mix the DNA with the buffer solutions.
- Set micropipettor to 80 microliters.
- Place glass slide smooth-side down, and rough-side up.
- Using the micropitettor place an 80 microliter drop of SYBR GREEN 1 on the slide between two dots.
- Using a different pipet tip, add 80 microliters of the DNA buffer solution to the 80 microliter dot of the SYBR GREEN 1.
- Align the drop with the LED light on the Fluorimeter, making sure that the light is focused on the drop.
- Place the black box over the Fluorimeter and camera set up. Then using the settings on the camera that where previously described focus on the drop and take the picture, being sure to close the flap of the box slowly before the picture is taken to ensure good photo quality. Take three pictures.
- Remove the box, and using the micropipettor remove the drop from the slide and discard it in a waste cup. Remove the slide from the Fluorimeter and discard it in a waste cup.
- Repeat steps 5-10 for the other concentrations of calf thymus solution, being sure to change the tip of the pipet with each new solution.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High:
Low:
Zero:
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
|
'
|
'
|
MEAN
|
|
|
|
Image 1 |
Image 2 |
Image 3 |
|
| 5 |
2.5 |
C-1 |
7347447 |
7406131 |
7755315 |
7551381
|
| 2 |
1 |
C-2 |
7238740 |
9035879 |
8643277 |
7941008.5
|
| 1 |
0.5 |
C-3 |
8152936 |
7891067 |
8093272 |
8123104
|
| 0.5 |
0.25 |
C-4 |
4583234 |
4477540 |
4285141 |
4434187.5
|
| 0.25 |
0.125 |
C-5 |
4035125 |
4276623 |
4233111 |
4134118
|
| 0 |
0 |
C-6 |
3162301 |
2502419 |
3046180 |
3104240.5
|
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control
Positive 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)
|
"
|
| + |
8960282.667 |
1.039717333 |
12 |
12.476608
|
| - |
4516405.333 |
5.483594667 |
12 |
65.803136
|
| 1_1 |
3860188.333 |
6.139811667 |
12 |
73.67774
|
| 1_2 |
4322908 |
5.677092 |
12 |
68.125104
|
| 1_3 |
4207222.667 |
5.792777333 |
12 |
69.513328
|
| 2_1 |
5477488.667 |
4.522511333 |
12 |
54.270136
|
| 2_2 |
4274024 |
5.725976 |
12 |
68.711712
|
| 2_3 |
4120281.667 |
5.879718333 |
12 |
70.55662
|
|
|
PCR Results: Summary
- Our positive control PCR result was 12.476608 μg/mL
- Our negative control PCR result was 65.803136 μg/mL
Observed results
- Patient 64291 : The image obtained from this patient closely resembled the image of our negative control, in that the drop appeared to be extremely clear with little to no highlighting after manipulation.
The μg/mL for each of the three trails conducted for this patient where as follows:
Trial 1: 73.67774 μg/mL, Trial 2: 68.125104 μg/mL, Trial 3: 69.513328 μg/mL
Which were similar to that of our negative control which was 65.803136 μg/mL
- Patient 76291 : The image obtained from this patient closely resembled the image of our negative control, in that the drop appeared to be extremely clear with little to no highlighting after manipulation.
The μg/mL for each of the three trails conducted for this patient where as follows:
Trial 1: 54.270136 μg/mL, Trial 2: 68.711712 μg/mL, Trial 3: 70.55662 μg/mL
Which were similar to that of our negative control which was 65.803136 μg/mL
Conclusions
- Patient 64291: Patient 64291's PCR results showed close resemblance to that of our negative control. Therefore our final conclusion for Patient 64291 is negative.
- Patient 76291: Patient 76291's PCR results also showed close resemblance to that of our negative control. Therefore our final conclusion for Patient 76291 is negative.
|
BME 100 Fall 2015
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