OUR TEAM
LAB 5 WRITE-UP
PCR Reaction Report
The pre-lab helped refresh our memory on the exact procedures needed when using a micropipette. Our experience with using a micropipette was excellent and we fully understand the difference between the first and second stop of the micropipette. There was one point where there was a little bit of the liquid left on the tube, but not too bad. Overall, the use of micropipettes was success.
Fluorimeter Procedure
Smart Phone Camera Settings
- Type of Smartphone: Samsung Note3
- Flash: Off
- ISO setting: Maximum
- White Balance: Automatic
- Exposure: 0
- Saturation: N/A
- Contrast: N/A
Camera set-up
- We gathered a phone holder/stand and vertically inserted the phone. Using a plastic box we stabilized the phone. We then measured thirteen centimeters between the phone and the fluorimeter.
- Distance between the smart phone cradle and drop = 13 centimeters
Placing Samples onto the Fluorimeter
- Step one: Turn on the Fluorimeter
- Step two: While wearing gloves place the smooth side of the slide down on the Fluorimeter
- Step three: Place the camera 13 centimeters away from the Fluorimeter
- Step four: Adjust camera to line up the picture
- Step five: Pipette 80 microliters of SYBR green on the slide in between the 2 sets of dots
- Step six: Pipette 80 microliters of the sample with the 80 microliters of SYBR green
- Step seven: Move the slide so the light shines through the middle of the drop
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5mg/mL
0.5 mg/mL
ZERO 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
|
|
|
|
Image 1 |
Image 2 |
Image 3 |
|
|
| 5 |
2.5 |
C-1 |
26422 |
27830 |
27194 |
27148.66667 |
705.0938472
|
| 2 |
1 |
C-2 |
28838 |
29510 |
29510 |
29286 |
387.9793809
|
| 1 |
0.5 |
C-3 |
29178 |
27081 |
26528 |
27595.66667 |
1397.957915
|
| 0.5 |
0.25 |
C-4 |
32079 |
32458 |
31500 |
32012.33333 |
482.4669246
|
| 0.25 |
0.125 |
C-5 |
29451 |
31190 |
3614 |
21418.33333 |
15443.50169
|
| 0 |
0 |
C-6 |
28291 |
2991 |
13392 |
14891.33333 |
12716.46572
|
|
|
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Group
Negative Group
PCR Results: PCR concentrations solved
PCR Results: Summary
- Our positive control PCR result was 45.80738602 μg/mL
- Our negative control PCR result was 41.01719245 μg/mL
Observed results
- Patient 33309 : The image for the positive control group was dim at first sight. After comparing to the negative control group, a slight difference in the top stripe was noticed. This was confirmed through the initial finding of the ImageJ measurement of the RAWINTDEN Of the Drop of 1058630 which was much higher than the negative control results, as discussed below. The calculation of the positive control group's PCR result was 45.80738602 μg/mL, confirming the higher concentration.
- Patient 32924 : The image for the negative control results did show a slight stripe for the green image after analyzing the positive control group. But, the image showed noticeably less saturation than the positive control group at the measurement of RAWINTDEN of the Drop at the number 748912. The calculation of the negative control group's PCR result was 41.01719245 μg/mL, confirming a lower concentration.
While both images first appeared to have no difference between them, this was discounted through the measurements of ImageJ and the concentration levels through the calculations.
Conclusions
- Patient 33309 : Patient one results compared to positive and negative control value was inconclusive. Further data is required to determine whether the patient one is positive or negative.
- Patient 32924 : Patient two's results determined a positive result for the test. No further testing is necessary.
| 
BME 100 Fall 2015
