The objective of PCR lab 3 was to prepare DNA samples to be placed in the thermal cycler to conduct the PCR process. The materials required was the DNA samples from two different patient’s, a pipette, disposable pipette tips, PCR tubes, positive and negative controls, and eight tubes of primer solution to mix with the patient samples and the controls. First, the empty PCR tubes were separated into sections of four connected PCR tubes and Leah and Abby labeled with the patient’s information. While the PCR tubes were being labeled Nadia, Alejandra, and Oscar reviewed the lab workbook for the correct techniques to use while combining the multiple components for PCR.
Abby used the micropipette to first transfer 50 microliters of PCR reaction solution to the empty PCR tube. After discarding the pipette tip after the first transfer, Abby then transferred the 50 microliters of primer mix into the same PCR tube to make a total of 100 microliters. This process was repeated with the other seven PCR tubes with the end result being one positive and one negative control of PCR solution, three tubes containing the DNA of patient one, and three tubes that contained the DNA of patient two. Once all tubes had the DNA and the primers, the PCR tubes were placed in the thermal cycler to conduct the continuous heating and cooling process.
All members of group four properly discarded the pipet tips and the supplies used to transfer the controls, patient replicates, and the DNA primer mix.
Fluorimeter Procedure
Imaging set-up
The fluorimeter was placed upon a platform so that the smartphone camera would be aligned to take a picture from the side. The smartphone camera was set to take three photos with no flash on a five-second countdown timer so that the lightbox could be closed in time. When ready, the lightbox was placed over the fluorimeter so that when closed, a minimal amount of light could shine through the door and accurate photos could be taken of the sample.
Placing Samples onto the Fluorimeter
Step 1: An 80 μL drop of SYBR GREEN solution was placed upon the glass slide near the front, in between the two right rows so that the drop was round and stabilized.
Step 2: 80 μL of the sample solution was added to the drop of SYBR GREEN, making sure the full drop was stabilized in between the two dots.
Step 3: The glass slide was moved so that the blue LED light was focused through the solution drop onto the black fiber optic on the left side of the fluorimeter.
Step 4: A light box was placed over the fluorimeter. The smartphone was placed on the cradle so that the side of the drop was in focus. A timer was set to take three photos, and the light box was closed for this time.
Step 5: All steps were repeated for all concentrations of calf thymus DNA and the PCR samples.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 micrograms/mL Calf Thymus DNA
.5 micrograms/mL Calf Thymus DNA
0 micrograms/mL Calf Thymus DNA (pure water)
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
16422500
14444505
12995204
14620736.33
1720430.929
2
1
C-2
14663491
13546174
12172894
13460853
1247488.721
1
0.5
C-3
12075274
12801666
12643553
12506831
382009.1797
0.5
0.25
C-4
7734444
8847778
8855907
8479376.333
645143.1284
0.25
0.125
C-5
3867222
4753889
4427953.5
4349688.167
448484.8816
0
0
C-6
649631
421687
1566481
879266.3333
605960.0774
Calibration curves
Images of Our PCR Negative and Positive Controls
Negative Control PCR Sample
Positive Control PCR Sample
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
RAWINTDEN DROP - BACKGROUND
Mean
Standard Deviation
Image 1
Image 2
Image 3
G4-neg
627331
616586
618780
620899
5677.268445
G4-pos
729022
752644
725177
735614.3333
14872.9004
G4-1 (1)
10177169
9705804
10150462
10011145
264770.0147
G4-1 (2)
3223111
3305722
3139767
3222866.667
82977.7698
G4-1 (3)
16391910
15389034
15237078
15672674
627493.4057
G4-2(1)
8727631
8384113
8877497
8663080.333
252946.7025
G4-2 (2)
7786897
6731392
7217747
7245345.333
528293.4339
G4-2 (3)
4769311
4908953
4269998
4649420.667
335925.7984
PCR Results: Summary
Our positive control PCR result was -4.529 μg/mL
Our negative control PCR result was -4.758 μg/mL
Observed results
Patient 46547 : The images showed high levels of green fluorescence. The concentration levels found for the three samples (in μg/mL) were 14.022, .446, and 25.35.
Patient 41967 : None of the images exhibited any green fluorescence. The concentration levels found for the three samples (in μg/mL) were 11.32, 8.49, and 3.30.
Conclusions
Patient 46547 Positive, due to fluorescence observed and higher mean concentration value (like the positive control).
Patient 41967 Negative, due to lack of fluorescence and lower mean concentration value (like negative control).