OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab reading allowed the experiment to proceed with ease. Additionally, it
helped the team understand the difference between the first stop and the second stop on the micropipette. The first stop is used when retrieving the sample; the second stop is used when inputting the sample into the designated tubes. The final reactions all had the same amount of liquid (100 microliters) once the DNA and the PCR reaction mix were added to each tube. The labeling scheme matched that of the one mentioned in lab report 4.
Fluorimeter Procedure
Imaging set-up
1. Go into camera menu.
2. Turn off flash.
3. Change ISO to 800.
4. Change White Balance to Auto.
5. Change exposure to highest option.
6. Change saturation to the highest setting.
7. Change contrast to the lowest setting.
Placing Samples onto the Fluorimeter
1. Elevate the fluorimeter until a mobile phone can be placed in a suitable position to picture the slide and drop of liquid.
2. Put 80 microliters of SYBR GREEN and 80 microliters of one of the calf thymus solutions or water onto the slide so it forms a drop.
3. Turn on the blue LED and align the drop with the light so it focuses on the black fiber optic on the other side of the drop.
4. Place the camera in the stand and focus on the drop.
5. Take three images of the drop and be careful to focus the image properly.
6. Clean the slide and repeat the steps with the other concentrations of calf thymus DNA.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
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 |
Standard Deviation
|
| 5 |
2.5 |
C-1 |
552637 |
552637 |
0
|
| 2 |
1 |
C-2 |
693915 |
693915 |
0
|
| 1 |
0.5 |
C-3 |
620752 |
620752 |
0
|
| 0.5 |
0.25 |
C-4 |
569323 |
569323 |
0
|
| 0.25 |
0.125 |
C-5 |
409343 |
409343 |
0
|
| Water |
0 |
C-6 |
371801 |
371801 |
0
|
Calibration curves
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Images of Our PCR Negative and Positive Controls
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PCR Results: PCR concentrations solved
| PCR Product TUBE LABEL |
MEAN (of RAWINTDEN DROP-BACKGROUND) |
PCR Product COncentration (ug/mL) |
Total Dilution |
Initial PCR Product Concentration (ug/mL)
|
| G4+ |
605084 |
5.802 |
12 |
69.624
|
| G4- |
281714 |
-4.863 |
12 |
-58.356
|
| G4 1-1 |
706874 |
9.159 |
12 |
109.908
|
| G4 1-2 |
530104 |
3.329 |
12 |
39.948
|
| G4 1-3 |
454130 |
0.823 |
12 |
9.876
|
| G4 2-1 |
262392 |
-5.5 |
12 |
-66
|
| G4 2-2 |
363067 |
-2.18 |
12 |
-26.16
|
| G4 2-3 |
374371 |
-1.807 |
12 |
-21.684
|
PCR Results: Summary
- Our positive control PCR result was 69.624 μg/mL
- Our negative control PCR result was 58.356 μg/mL
Observed results
- Patient 1 : (49532) We observed a green color in the drop for patient one.
- Patient 2 : (15765) There was no green color present in the drop for patient two.
Conclusions
- Patient 1 : (49532), Patient 1's results were very similar to the positive control value and were very different from the negative control value. Therefore, Patient 1 was positive due to the fact that Patient 1's results were very similar to the positive control value.
- Patient 2 : (15765), Patient 2's results were very similar to the negative control value and were very different from the positive control value. Therefore, Patient 2 was negative due to the fact that Patient 2's results were very similar to the negative control value.
| 
BME 100 Fall 2018
