In our PCR reactions test, we were given DNA samples from two different patients. With three samples from each, we were able to test for diseases with PCR reaction mix and primers. We took each sample of DNA, along with our positive and negative controls and individually mixed PCR reaction mix and primers to them. Then 8 samples we had mixed together were placed in a thermocycler to amplify the DNA. After the samples ran through the thermocycler the positive control and samples with disease should have a high frequency of DNA because the primer was able to attach to disease DNA sections. While the other samples should have a low frequency because there was no disease DNA sections for the primer to attach to. Overall the procedure went well, and it gave our team the chance to have extra experience using a micropipette, learning correct form, and give us a better understanding of the lab we were completing.-
Fluorimeter Procedure
Imaging set-up
1. Changing Camera Settings
Set Flash to OFF
ISO 800
Set exposure at it's highest setting
Set saturation at it's highest setting
Set contrast at lowest settings
Camera timer set to 3 seconds
2. Place the camera in the slot, allowing the drop on the fluorimeter slide is focused. (Make sure the camera is as close to the drop as possible.)
3. Maintain the distance between the camera and fluorimeter (measuring before each picture).
4. Make sure you cover the fluorimeter and camera with a box to ensure the picture is taken in complete darkness.
Placing Samples onto the Fluorimeter
Turn the fluorimeter on. Make sure the blue light is on.
With gloves on, place the fluorimeter in place. Make sure the smooth side is faced down.
Take the micropipette and set it to 80 micro liters
Place a new pipette tip on and push down to the first stop
"Insert the clean tip into the SYBR Green I solution and release the pipette plunger slowly until all 80 micro liters are inside the pipette"
"Release all 80 micro liters of SYBR Green I solution on the very first 2 circles in the middle of the fluorimeter slide. To release the solution, push down on the micropipette until you feel the second stop"
"Replace the tip of the micropipette
"Collect 80 micro liters of the sample/calibration solution over the drop of SYBR solution"
"After taking pictures of the drop, take the drop off of the slide with the micropipette and discard the tip"
"Adjust the slide to the next two circles and repeat these steps for the rest of your solutions"
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Image 1: 5 μg/mL sample
Image 2: 0.5 μg/mL sample
Image 3: 0 μg/mL 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
9323706
12451442
11604547
11126565
1617724.703
2
1
C-2
573747
409934
350329
444670
115688.5602
1
0.5
C-3
344314
344437
343444
344065
541.3067522
0.5
0.25
C-4
446416
449778
448296
448163.3333
1684.921759
0.25
0.125
C-5
390945
394866
403998
396603
6697.617412
0
0
C-6
7983234
9339861
7429156
8250750.333
983042.2884
Calibration curves
Graph 1: Calibration Curve
Graph 2: Calibration Curve (without the highest concentration 5 μg/mL)
Images of Our PCR Negative and Positive Controls
Image 4: Negative control PCR sample
Image 5: Positive control PCR sample
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)
G8+
17684747
13.83196089
12
165.9835307
G8-
9350946.667
1.1682467
12
14.0189604
G81-1
14657524.67
8.383190629
12
100.5982876
G81-2
12731779.67
4.916995903
12
59.00395083
G81-3
13293115
5.927356866
12
71.12828239
G82-1
7501006
4.497999384
12
53.97599261
G82-2
14138896
7.449698423
12
89.39638107
G82-3
10584719.33
1.052450387
12
12.62940464
PCR Results: Summary
Our positive control PCR result was 165.9835307 μg/mL
Our negative control PCR result was 14.0189604 μg/mL
Observed results
Patient 16653 : Patient 1(16653) was tested multiple times. The three results for initial PCR product concentration showed 100.5982876, 59.00395083 and 71.12828239. The tested sample with a higher mean had an initial concentration closer to the positive control's initial concentration, showing that the patient may test positive. Looking at the pictures of the drops on the fluorimeter, it seems to follow closely to positive. But the other samples say otherwise.
Patient 14852 : Patient 2(14852) was tested multiple times as well. The three results for initial PCR product concentration showed 53.97599261, 89.39638107, and 12.62940464. The tested sample with a higher mean had an initial concentration close to the negative control's initial concentration. This now shows that this patient is testing positive. Looking at the pictures of the drops on the fluorimeter, it seems to follow closely to negative. But again the other samples tested weren't so clear.
Conclusions
Patient 16653 : Even though some of the samples weren't close to the same initial concentration value as the positive, Patient 16653 would be tested positive. The sample with a higher concentration shows similar values to the positive control.
Patient 14852 : Again, some of the other samples weren't as consistent, but Patient 14852 would be tested negative. Just like the the positive sample, the negative control sample was closely related to the the sample with a higher mean.
BME 100 Spring 2018
