Our group felt well prepared while micropipetting and the pre-lab readings and videos gave us all a proper understanding of how to use the materials. Additionally, a member of our group had experience with micropipettes and was able to provide insight and advice while we performed the lab. We understood the difference between the first and second stop and we were able to use them with relative ease. The final reaction retained the same amount of liquid because we used the pipette to extract all of the liquid in the samples that we had. There was no liquid left in the tubes of DNA samples or the PCR reaction mix. We did not change the label scheme, because it was familiar to us and we saw that it was the most efficient.
Fluorimeter Procedure
Imaging set-up
The first part of the setup was to ensure that the proper PPE was put on. Then the following materials were gathered. A fluorimeter, 3 glass slides, micropipette, pipette holding box, iPhone 6(s).
The camera was first set up to find the proper distance and height of the fluorimeter. The iphone was placed into the phone stand and was placed 7.5cm away from the fluorimeter. The fluorimeters height was adjusted by stacking small white boxes underneath it. The height was made so that the camera was directly aligned with the glass slide and had an optimal view of the beam of light. The fluorimeter was turned on and a slide was inserted so that the beam of light crossed directly through two of the circles on the slide. The phone was set on a timer of 3 seconds to take an image, this would occur three times for each sample. The phone was focused on the sample in the slide and the fluorimeter cover was placed on the whole set up. The images were taken while the cover was on and the sample was in darkness.
Placing Samples onto the Fluorimeter
Insert slide into fluorimeter with the rough siding facing upward
The micropipette dial was set to 80 µL
Extract the SYBR Green I
Place the SYBR Green I on the slide in between the two circles where the beam of light shines across
Repeat steps 3 and 4 for the sample
place the sample on top of the SYBR Green I
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Calf Thymus DNA Image
Low Calf Thymus DNA Image
Zero Calf Thymus DNA Image
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
219957
222535
231709
224734
6176.80964
2
1
C-2
237898
251665
263075
250879
12606.87457
1
0.5
C-3
248869
435608
261913
315463
104252.5404
0.5
0.25
C-4
396062
239558
273599
303073
82309.85974
0.25
0.125
C-5
242373
320502
292651
285175
39597.34024
0
0
C-6
387280
251162
311667
316703
68198.59568
Calibration curves
Images of Our PCR Negative and Positive Controls
PCR Negative Control Image
PCR Positive Control Image
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)
G9+
8454332
-461.735
12
-5540.823
G911
11865515.6
-655.091
12
-7861.093
G912
15228216.6
-845.699
12
-10148.385
G913
21831970
-1220.019
12
-14640.225
G9-
4732420.66
-250.766
12
-3009.197
G921
5835298
-313.281
12
-3759.369
G922
4973007.33
-264.404
12
-3172.843
G923
4122233.66
-216.179
12
-2594.151
PCR Results: Summary
Our positive control PCR result was -55540.823 μg/mL
Our negative control PCR result was -3009.197 μg/mL
BME 100 Fall 2017
