Due to the pre-lab materials, and some of our team member’s previous experience, our team was well informed before the lab of proper pipetting technique. To begin the lab we gathered our materials and properly labeled the sides of the PCR tubes for our group, following our template from part A of the lab. Our tubes were labeled as: G1 +, G1 -, G1 1-1, G1 1-2, G1 1-3, and G1 2-1, G1 2-2, G1 2-3, to show the different trials for each patient, along with our positive and negative controls. Before we began pipetting, we made sure our samples were fully thawed, in order to get correct measurements from the pipetting. Our pipetting was done in a group of two, for continuous quality check. We had some volume issues with our pipetting, and with the assistance of Dr. Haynes and some extra aster mix, these issues were corrected, giving us the proper volume of 50 µL in each PCR tube. After they were all properly pipetted, we programmed the PCR machine in accordance to the Lab Workbook. We then notified a TA, and they helped us load the tubes into the PCR machine, and have it properly programmed.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: iPhone (back-up), Samsung Galaxy Note 4 (used)
Flash: off
ISO setting: No setting, Set to 800
White Balance:No setting, Auto
Exposure: No setting, +2: highest setting
Saturation: No setting, No setting
Contrast: No setting, -2: lowest
Camera set-up
We placed the fluorimeter on top of two empty pipette tip trays, and then positioned the camera 8 cm away from the flourimeter, adjusted in place by folded paper, in order to keep the phone straight up so we can take a sideways picture. The camera timer was set at 5 seconds. We marked the location and remeasured the distance when we moved the camera between trials
Distance between the smart phone cradle and drop = 8 cm
Placing Samples onto the Fluorimeter
Attach a new pipette tip to the micropipetter, which is set at 80 μL
Add 80 μL of SYBR Green I Solution in a single drop between the middle circles in the first two rows on the rough surface of a new slide
With a new pipette tip, add 80 μL of either the sample or blank onto the previously present drop of SYBR Green I
Align blue light in the fluorimeter with the center of the drop
Place the fluorimeter under an upside down box that blocks out all other light
Place the camera with the above-mentioned settings 8 cm in front of the fluorimeter
Press the capture button, thus activating the timer, and close the flap
Wait for the camera to capture the picture and repeat steps 6-8 for a total of 3 pictures
Remove the slide and discard all material on it
Repeat steps 1-9 for each sample needing testing (in this case, 6 times for calibration and 8 times for PCR reactions)
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
High Concentration:
Low Concentration:
No Concentration:
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
11500687
11727326
870850
8032954.333
6203599.372
2
1
C-2
12763552
13260820
10054390
12026254
1725689.599
1
0.5
C-3
9120081
9101978
9790216
9337425
392232.9626
0.5
0.25
C-4
7612011
8115471
8292500
8006660.667
353052.5262
0.25
0.125
C-5
3082232
2782889
3201476
3022199
215654.2139
0
0
C-6
3401514
2545859
2851382
2932918.333
433615.6132
Calibration curves
Images of Our PCR Negative and Positive Controls
Positive Control:
Negative Control:
PCR Results: PCR concentrations solved
PCR Product TUBE LABEL
RAWINTDEN DROP - BACKGROUND
'
'
MEAN
Standard Deviation
Image 1
Image 2
Image 3
G1 +
11197613
10004860
9616318
10272930.33
824026.5451
G1 -
2557014
2444811
2511807
10175677.33
56453.00416
G1 1-1
3712743
4079116
4283647
4025168.667
289250.0309
G1 1-2
2879984
3237511
3787064
3301519.667
456915.0508
G1 1-3
3650829
3136043
3450691
3412521
259506.9712
G1 2-1
13790299
15301484
16786776
15292853
1498257.145
G1 2-2
9892540
10560817
9130519
9861292
715660.8277
G1 2-3
11688632
12419617
11616580
11908276.33
444297.0073
PCR Results: Summary
Our positive control PCR result was 2.136 μg/mL
Our negative control PCR result was 2.087 μg/mL
Observed results
Patient 30522 : For our first patient, the images from the fluorimeter gave us all images without the green glow,and after calculating the data, we determined that all of the patient's values were below 2.087
Patient 46645 : Our second patient's pictures all appeared a vibrant green, and 2 of the 3 values given were about the positive control limit of 2.136
Conclusions
Patient 30522 : All three of the patients trials were below the lower bound of 2.087, and the pictures did not have a green tone, we determined finally that our patient 30522 is negative.
Patient 46645 : Our second patient had the majority of his values significantly over the higher bound of 2.136, and in our experiments with the fluorimeter, all of the images appeared to have the green glow. Therefore, with the information, we determined that our patient 46645 is positive.
BME 100 Fall 2015
