Name: Morgan Baerwaldt Role(s): Wet Lab Work, Excel Graphs/Analysis, Wiki Writeup
Name: Daniel Gaytan-Jenkins Role(s): Wet Lab Work, ImageJ Analysis, Excel Data Recording
Name: Cory Kehoe Role(s): ImageJ Analysis, Excel Data Recording
Name: Pedro Lopes Role(s): Wet Lab Work
Name: Brittany Metzler Role(s): ImageJ Analysis, Excel Data Recording
Note: One team member, Jaxon Lewandowski, was not present for this lab.
LAB 5 WRITE-UP
PCR Reaction Report
Group 12 did not have very much trouble with pipetting the samples. The pre-lab reading was helpful, and we quickly watched the provided YouTube video before starting to make absolutely sure that we knew what to do. Finding the first and second stop while pipetting was not an issue, and our final reactions had very similar amounts of liquid. We had to go back to pick up liquid that was left behind a few times, but we were careful to retain as much of the liquid as we could and ended up losing little. There were no problems with the labeling scheme. We labeled our PCR tubes in the format G12 - [patient #] - [trial #], where patient number was 1 or 2, with 1 corresponding to patient 32901 and patient 2 corresponding to patient 69184. The positive and negative controls were labeled in the format G12 - [+ or -].
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: Samsung Galaxy S6
Flash: no
ISO setting: 800
White Balance: auto
Exposure: auto
Saturation: auto
Contrast: auto
Camera set-up
The phone was set up in the stand in portrait mode. The slide was raised closer to the camera with several plastic plates so that it was nearly level with the camera, with the camera very slightly above it in order to obtain the best possible view of the drop.
Distance between the smart phone cradle and drop = 9 cm
Placing Samples onto the Fluorimeter
Use a micropipette to draw an 80 mL sample of SYBR Green dye. Next, empty the micropipette into center of the slide to form a round drop.
Use a micropipette to draw an 80 mL sample of one of the calf thymus sample solutions. Next, carefully empty the micropipette onto the center of the SYBR Green drop so that the two solutions form one drop.
Align the slide so that the blue light passes directly through the dot and is focused onto the black fitting on the other side.
After images are taken, use the pipettor to fully remove the 160 microliter drop of solution.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High concentration (5 μg/mL sample):
Low concentration (0.5 μg/mL sample):
Zero DNA (0 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
MEAN
Standard Deviation
Image 1
Image 2
Image 3
5
2.5
C-1
1674974
1698265
1681049
1684762.7
12081.4
2
1
C-2
1689383
1684111
1669040
1680844.7
10557.5
1
0.5
C-3
1333423
1335537
1335732
1334897.3
1280.5
0.5
0.25
C-4
1209021
1228229
1209311
1215520.3
11007.0
0.25
0.125
C-5
725993
896159
893952
838701.3
97614.5
0
0
C-6
318549
320246
330640
323145.0
6546.1
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 (µg /mL)
Total Dilution
Initial PCR Product Concentration (µg /mL)
+
1713825.667
1.831
12
21.970
-
636813.000
-0.00179
12
-0.0215
1-1
586131.333
-0.0880
12
-1.056
1-2
554483.000
-0.142
12
-1.703
1-3
602059.333
-0.0609
12
-0.731
2-1
1725944.667
1.851
12
22.218
2-2
1771529.333
1.929
12
23.149
2-3
1794974.000
1.969
12
23.627
PCR Results: Summary
Our positive control PCR result was 21.970 μg/mL
Our negative control PCR result was -.0215 μg/mL
Observed results
Patient 32901 : These droplets were dark blue at the bottom and light at the top. They did not appear to be green or glowing. The blue light source could clearly be seen on both sides of the drop, but did not show up as well in the middle of the drop. The initial PCR product concentrations were calculated to be -1.056, -1.703, and -0.731 μg/mL, respectively, for each of the three trials.
Patient 69184 : These droplets were bright green at the bottom, fading to teal and light blue at the top. The light on the left (source) side of the drop was light blue, but the light shining through the other side of the dot was greenish blue. The initial PCR product concentrations were calculated to be 22.218, 23.149, and 23.627 μg/mL, respectively, for each of the three trials.
Conclusions
Patient 32901 : The calculated results are smaller than the concentration for the negative control. This evidence indicates that the patient is probably negative for the SNP, as the sample had a smaller amount of PCR product than a sample known to not contain the SNP. The fact that calculated PCR product concentration was negative indicates error in our trendline equation.
Patient 69184 : The calculated results are greater than the concentration for the positive control. This evidence indicates that the patient is probably positive for the SNP, as the sample had a greater amount of PCR product than a sample known to contain the SNP.
BME 100 Fall 2015
