Going through the pre lab material was not only extremely important to understand how to pipette, but it made the lab run smoothly. Doing the PCR virtual lab helped explain the reason why we are pipetting. The virtual micro-pipetting exercise familiarized us with the micro pipettor.We all practiced using the pipette with no liquid to understand the difference between the first and second stop. Practicing micro-pipetting allowed us to be more precise when mixing the PCR primer mixture and the DNA, and ensured that all the final reactions had the same amount of liquid. To double check that the amounts were the same, we checked to make sure there was no liquid left in the tubes that the DNA samples and PCR reaction mix was taken from. Since everything was properly labeled at the start of the lab, we did not have to change our labeling scheme.
Fluorimeter Procedure
Imaging set-up
It was important to put on all PPE before starting. Then all the materials were gathered. This included the fluorimeter, 3 glass slides, an iPhone 6, 3 small pipette holder boxes. First, one side of the fluorimeter box was opened and the lid was removed so everything could be easily set up. Then the smooth side of the slide was put face down in the fluorimeter. Next, the phone camera was turned on and set to a 3 second timer. It was then placed on the phone holder and an 80 microliters sample of H2O was pipetted on the first 2 clear circles in the middle of the slide. Then 3 small pipette holder boxes were placed beneath the fluorimeter to get the droplet level with the camera, and the camera distance was adjusted so a clear image of the droplet side could be taken. Once the camera was clearly focused on the H2O sample, the H2O was removed and the fluorimeter was ready for imaging. The lid of the fluorimeter was put on every time a picture was taken to prevent any source of light getting in.
Placing Samples onto the Fluorimeter
Set the micro pipette to 80 micro liters
Put on a clean pipette tip
Push plunger button to first stop
Insert tip into SYBR Green I solution
Slowly release plunger button until all 80 micro liters are inside the pipette
Place all 80 micro liters of SYBR Green I solution on first 2 clear circles in the middle of the slide by pushing plunger button all the way down to the second stopper
Eject pipette tip into discard cup
Put on new clean pipette tip
Push plunger button to first stop
Insert into sample/calibration solution
Slowly release plunger button until all 80 micro liters are inside the pipette
Place all 80 micro liters of sample/calibration solution over first drop
After the pictures have been taken, suck up the whole drop on the slide using pipette
Eject pipette tip into discard cup
Adjust slide to the center of the next two holes
Change slide after all five possible measurement positions have been used
Repeat steps 2-16 until all calibration solutions have been put on the fluorimeter
Repeat steps 2-16 until all DNA sample solutions have been put on the fluorimeter
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 (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
17926243
16631330
16348318
16968630
841302.8364
2
1
C-2
14445901
13255933
13720561
13807465
599725.0947
1
0.5
C-3
12373343
9617279
10469292
10819971
1411100.347
0.5
0.25
C-4
10345440
12265350
12057955
11556248
1053705.74
0.25
0.125
C-5
7173099
6753052
7109988
7012046
226504.5212
0
0
C-6
0
0
0
0
0
Calibration curves
Calf Thymus DNA Calibration Curve 1
Calf Thymus DNA Calibration Curve 2
Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
Negative Control
Positive Control
PCR Product TUBE LABEL
MEAN (of RAWINTDEN DROP-BACKGROUND)
PCR Product Concentration (µg /mL)
Total Dilution
Initial PCR Product Concentration
(µg /mL)
G11 1-1
6991641.667
0.49582
12
4.77994
G11 1-2
28544989
11.272495
12
135.2699
G11 1-3
7234025
0.6170125
12
7.404
G11 2-1
4515353
-0.7423235
12
-8.90788
G11 2-2
7005001
o.5025005
12
6.030006
G11 2-3
7731292.33
0.8656165
12
10.3877
G11 +
16592518.33
5.296259165
12
63.555
G11 -
5221622
-0.389189
12
-4.670
PCR Results: Summary
Our positive control PCR result was 63.555 μg/mL
Our negative control PCR result was -4.670 μg/mL
Observed results
Patient 43374: The patients results of the PCR Product were all positive and well above O. G11 1-2 however, seems to have way more concentration than the others so that may have been caused because an error in the lab. The pictures of their droplets all seem to relatively clear with little to no white shading in it.
Patient 37706:The droplets seem to have a darker shade especially in the lower region of the droplet. The data for this patient only had one concentration that was negative and the rest was positive. All the PCR product concentration are close to zero except for
Conclusions
Patient 43374 :They tested positive for the disease because the result are very similar to that of the positive. the mean is close to the positive.
Patient 37706 :They tested negative for the disease because they had results similar to the negative results
BME 100 Fall 2017
