The objective of this experiment is to test DNA from imaginary patients for disease markers using the PCR reaction process. The pre-lab reading helped the team understand the purpose and procedures of the experiment. In order to conduct a PCR reaction, a DNA sample, primers, taq polymerase, and deoxyribonucleotides are needed for the solution. The online pipetting simulation tutorial demonstrated the proper pipetting techniques. After setting the volume and attaching a tip, the pipette is pressed down to the first stop and vertically inserted into the solution. Releasing the button causes the pipette to draw the solution into the tip, and pressing it all the way causes the pipette to discharge all of the solution.
The preparations began by labeling the sides of two lines with four test tubes each. The labels that were used on the first strip were +, 1:1, 1:2, 1:3 and on the second -, 2:1, 2:2, 2:3 as well as G13 on each of the strips in order to be able to differentiate the samples from other groups. After this the strips were placed in a rack and a pipette was turned to 50 μL. The pipette was then used to transfer PCR reaction mix into the + labeled test tube and then a fresh pipette tip was put on the pipette. 50 μL of the positive control DNA/ primer mix was also transferred into the + labeled test tube. These two transfers of solutions were repeated for each of the test tubes but with the use of the respective DNA/primer mix. It was important to change the tip of the pipette in between every transfer to avoid cross-contamination. This led to all test tubes in the end containing DNA/primer mix and PCR reaction mix with a total of 100 μL of solution. The lids were then closed tightly and and the strips placed in a PCR machine. Finally, the PCR machine was started and the PCR reaction conducted.
Fluorimeter Procedure
Imaging set-up
To start the setup, a phone is held upright using a holder. Open the camera app on the phone and turn off flash function. The fluorimeter is placed exactly 5 cm away from the phone and raised so that the camera is on the same level as the fluorimeter. Turn on the fluorimeter and insert the glass slide with the smooth side down. Place a solution of 80 mL of the sample in between the first two row of dots on the slide, and then add 80mL of SYBR Green dye on top of the sample. A blob should form in the middle; adjust the slide till the fluorescent light shines directly through it. Make sure that the phone camera can focus on the blob.
To capture fluorescence from the sample through an image, the photos must be taken under specific conditions. There can be no external light, and the photos need to be taken from the same angle and distance each time. Place the light box over the fluorimeter and the phone, but keep one of the flaps open so that the camera can be accessed. Take the photo by setting a 3 second timer on the camera, and lowering the flap before the picture is taken. Repeat this three times for each sample.
Placing Samples onto the Fluorimeter
Turn on the fluorimeter
Insert the glass slide into the fluorimeter with the smooth side down
Adjust the slide so that the fluorescent light goes in between the first two row of dots
Set a micropipette to 80 µL and attach a new tip
Extract 80 µL from the sample DNA and slowly eject the solution in between the first two row of dots on the glass slide
Dispose the tip properly, and replace it with a new tip
Extract 80 µL from the SYBR Green dye and eject the solution directly on top of the sample DNA on the slide
align the solution on the glass slide to the fluorescent light so that the light shines through
After taking the photos, remove the solution by using the micropipette
Dispose both the tip and the solution in proper waste containers
Move the slide down to the next two rows of dots
Repeat steps 4 to 8 for the other samples
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL sample
0.5 μg/mL sample
zero DNA
Calibrator Mean Values
Initial Concentration of 2X Calf Thymus DNA solution (µ/mL)
Final DNA concentration in SYBR Green I Solution (µ/mL)
Sample Number
RAWINTDEN DROP-BACKGROUND: Image 1
RAWINTDEN DROP-BACKGROUND: Image 2
RAWINTDEN DROP-BACKGROUND: Image 3
MEAN
Standard Deviation
5
2.5
C-1
1153684
1229732
1138372
1173929.333
48929.20949
2
1
C-2
959603
950900
944221
951574.6667
7713.16163
1
0.5
C-3
788053
759645
757349
768349
17102.73709
0.5
0.25
C-4
655481
666213
660358
660684
5373.421908
0.25
0.125
C-5
647474
600633
649644
632583.6667
27691.35335
0
0
C-6
311327
358302
301742
323790.3333
30269.77714
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
MEAN (of RAWINTDEN DROP-BACKGROUND)
PCR Product Concentration (µ/mL)
Total Dilution
Initial PCR Product Concentration (µ/mL)
G13 +
606073
0.516744009
12
6.20092811
G13 1-1
670938
0.763472803
12
9.16167364
G13 1-2
620790
0.572723469
12
6.872681628
G13 1-3
545198
0.285192088
12
3.422305059
G13 2-1
309486
-0.611392164
12
-7.336705972
G13 2-2
307903
-0.617413465
12
-7.408961582
G13 2-3
401907
-0.259847851
12
-3.118174211
G13 -
391260
-0.300346139
12
-3.604153671
PCR Results: Summary
Our positive control PCR result was 6.20 μg/mL
Our negative control PCR result was -3.60 μg/mL
Observed results
Patient 97445 : The images of the droplets for this patient appeared to glow in a bright green color in the flourimeter. When the images were split using ImageJ, the droplet appeared white in the green channel, similar to the positive control sample. The test tubes for this patient are labeled as 1-1, 1-2, 1-3. The initial PCR product concentration of each test tube is 9.16 μg/mL, 6.87 μg/mL, and 3.42 μg/mL respectively.
Patient 71471 : The images of the droplets for this patient appeared to be more clear and transparent in the flourimeter. When the images were split using ImageJ, the droplet appeared gray in the green channel, similar to the negative control sample. The test tubes for this patient are labeled as 2-1, 2-2, 2-3. The initial PCR product concentration of each test tube is -7.34 μg/mL, -7.41 μg/mL, and -3.12 μg/mL respectively.
Conclusions
Patient 97445 : With the results from the lab, it can be concluded that this patient tested positive for the disease SNP. The three test tubes from this patient had an initial PCR product concentration close to the positive control sample, 6.20 μg/mL. Test tube 1-3 had a concentration of 3.42 μg/mL which is lower than the positive control and the other two test tubes for this patient. However when considering the average of the concentration of test tubes 1-1, 1-2, 1-3, it indicates a positive test for the disease SNP.
Patient 71471 : With the results from the lab, it can be concluded that this patient tested negative for the disease SNP. All three test tubes from this patient were close to or had even lower concentrations than the negative control sample, -3.60 μg/mL. Test tubes 2-1 and 2-2 had much lower concentrations of -7.34 and -7.41 μg/mL. Test tube 2-3 had a concentration of -3.11 μg/mL which is slightly higher than the negative control sample, but negligbly so. Overall, the concentrations from the test tubes of this patient indicate a negative test for the disease SNP.
BME 100 Fall 2017
