Overall, the process of pipetting the samples was a decently simple and quick task. The work was divided up between each of the team members as we followed the steps found in the workbook. When following the pre-lab reading, in addition to an in-class explanation, the process went smoothly. They were both very helpful in making sure the procedure went correctly and whatever was not understood by one member, got explained by another. For example, a majority of the group understood how to use a micropipettor as well as the difference between the first and second stop (collect sample vs. release it), but was effectively explained to those who didn't.
Each of the final reactions had the same amount of liquid but had excess liquid in some of the DNA sample tubes. This was due to being given too much of it to work with initially. There was no need to change the labeling of the tubes as each of the amounts were correct.
Fluorimeter Procedure
Imaging set-up
To obtain the images from the fluorimeter, we utilized an iPhone 8 Plus to take the photos. The phone was placed in a holder to maintain its position and the sensor was boosted by plates to set it up parallel to the phone camera. This meticulous positioning is what allows the camera to take an effective photo of the droplets to be properly analyzed. The flash on the camera was disabled and a timer was placed on as well to allow extra time to further darken the setting. The darkening is done in order to get rid of any outside light that could ultimately affect the SYBR Green I. To do this, a black box was utilized to be placed on top of the camera and sensor. When the droplets are on the sensor, the blue LED light is on, and the phone camera is placed, the photo is taken. While the photo timer is going, place the black box over the devices to get an ideal image to analyze.
Placing Samples onto the Fluorimeter
Placing the samples on the Fluorimeter is a fairly simple procedure.
Set the micro-pipette to 80 micro liters.
Place the slide rough side up in the Fluorimeter.
pipette 80 micro liters of SYBR green and place it on the slide in a bubble.
pipette 80 micro liters of the sample(either calibration or PCR products) and place it on top of SYBR green.
Set the phone to take pictures on a timer and take 3 pictures of each sample tested.
repeat steps 3 through 5 till all the samples are analyzed.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Test 0 μg/mL sample
Test 0.5 μg/mL sample
Test 5 μg/mL sample
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
Image 1
RAWINTDEN DROP - BACKGROUND
Image 2
RAWINTDEN DROP - BACKGROUND
Image 3
MEAN
Standard Deviation
5
2.5
C-1
21120229
25878958
19520162
22173116.33
3307567.149
2
1
C-2
16780867
15193591
15991140
15988532.67
793641.2122
1
0.5
C-3
21125992
22065214
20492738
21227981.33
791183.6454
0.5
0.25
C-4
15242756
15623929
15091887
15319524
274202.7841
0.25
0.125
C-5
15610117
14160460
16894630
15555069
1367915.973
0
0
C-6
11202188
8634685
9842398
9893090.33
1284501.926
Calibration curves
Images of Our PCR Negative and Positive Controls Positive Control
Negative Control
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP - BACKGROUND)
PCR Product Concentration (µg /mL)
(Step 5 calculation)
Total Dilution
Initial PCR Product Concentration,(µg /mL)
(Step 6 calculation)
G15-
7294286.33
-1.352856835
6
-8.11714101
G15+
15529445
2.7647225
6
16.588335
G15 1-1
3905329
-3.0473355
6
-18.284013
G15 1-2
10713300.33
0.356650165
6
2.13990099
G15 1-3
7792050
-1.103975
6
-6.62385
G15 2-1
7996279
-1.0018605
6
-6.011163
G15 2-2
7699558
-1.150221
6
-6.901326
G15 2-3
8597099.67
-0.701450165
6
-4.20870099
PCR Results: Summary
Our positive control PCR result was 16.588335 μg/mL
Our negative control PCR result was -8.11714101 μg/mL
Observed results
Patient 40686 : For this patient two out of the three pictures looked closely to our negative control which had zero showing of the SYBR Green 1. These pictures looked like water droplets in light making it clear enough to see through the droplet to the other side. For the results two came out to be negative, approximately -18 μg/mL and -6 μg/mL. The sample 1-2 came was a little less clear in the image and ended up being approximately positive 2 μg/mL.
Patient 68520 : This patient had very clear results on all three samples. All three samples had pictures that were very clear and also looked like pure water. There was an ability to see through them without any cloudiness/green from the SYBR Green 1. For the quantitative results samples 2-1 and 2-2 came out to be approximately -6 μg/mL as well. The other sample 2-3 was a little lower at -4 μg/mL.
Conclusions
Patient 40686 : This patient has been concluded to be negative for disease. This decision was made based of samples 1-1 and 1-3 because sample 1-2 was the odd one out. We figured that 1-2 results were due to human error or other limitations of this experiment. However since one was close to our negative control only being 2 μg/mL off and the other was significantly negative we concluded the patient to be negative.
Patient 68520 : This patient has been concluded to also be negative for disease. We used all three samples to conclude this. Two of the three were within 2 μg/mL of our negative control making it easy to conclude. The other sample was only within 4 μg/mL however it was still a negative number. This third sample also could have been a little off because of limitations. With all three of these being a negative number we decided this patient is also negative.
BME 100 Fall 2018
