The PCR reaction was a success. After collecting the PCR tubes, micropipetter, pipette tips, DNA samples, and enzyme mix, the PCR tubes were cut so that our team had 2 sets of 4 PCR tubes attached to one another. Then, the sides of the PCR tubes were labeled according to the following table.
Tube Label
PCR Reaction Sample
Patient ID
G13 +
Positive control
none
G13 -
Negative control
none
G13 1-1
Patient 1, replicate 1
24231
G13 1-2
Patient 1, replicate 2
24231
G13 1-3
Patient 1, replicate 3
24231
G13 2-1
Patient 2, replicate 1
48862
G13 2-2
Patient 2, replicate 2
48862
G13 2-3
Patient 2, replicate 3
48862
Having studied the pre-lab reading and completed the online tutorial regarding the use of a micropipetter, our team was quick and efficient at using the micropipetter for the PCR reaction. Each member understood the process of: Put on new pipette tip, depress plunger to stop 1, allow tip of pipette to submerge in the liquid, slowly allow plunger to return to the top, after 1 second delay remove the tip from the liquid, transfer tip into receptor, depress plunger to the second stop, hold, withdraw from solution, release plunger slowly, and eject tip into biological waste receptacle.
After transferring the DNA samples, positive and negative controls, and DNA/Primer mix into the assigned PCR tubes, the level of the liquid was observed. Upon visual inspection, most PCR tubes contained an equal amount of liquid. There was no excess liquid left in the tubes that the DNA samples came in, but there did appear to be a small amount of liquid left in the DNA/Primer mix tube.
Fluorimeter Procedure
Smart Phone Camera Settings
Type of Smartphone: LG G3
Flash: OFF
ISO setting: N/A
White Balance: N/A
Exposure: High
Saturation: N/A
Contrast: N/A
Camera set-up
The camera was propped so that the camera was upright in the cradle. Due to the high position of the camera due to the upright posture, the fluorimeter had to be elevated. A plastic tray was placed under the fluorimeter, making it even with the camera lens.
Distance between the smart phone cradle and drop = 12 cm
Placing Samples onto the Fluorimeter
The dark room flap was opened to give access to the fluorimeter.
A new glass slide was placed in the fluorimeter with the rough hydrophobic side facing up.
A new tip was affixed to the micropipetter, the plunger was depressed to the first stop and placed in the SYBR Green vial.
Releasing the plunger allowed a precise measured amount of SYBR Green to enter the micropipette tip.
The tip of the micropipetter was then moved to the glass slide in the fluorimeter, and the plunger was depressed slowly to the second stop, forming an drop on the slide.
The micropipette tip was ejected into the bio-waste receptacle. Then, a new tip was affixed, and a precise measured amount of the DNA sample was taken up.
The tip was placed in the SYBR Green drop formed on the glass slide, and the plunger was depressed to the second stop to allow the entire amount of DNA sample to coalesce with the SYBR Green drop.
The micropipette tip was ejected into the bio-waste container, and the slide was moved so that the beam of light passed straight from where it entered the drop to the other side of the fluorimeter.
The photo timer was primed, the dark room flask was closed, and a photo of the sample placed in the fluorimeter was taken.
Data Collection and Analysis
Images of High, Low, and Zero Calf Thymus DNA
Calibrator Mean Values
Final DNA concentration in SYBR Green I solution (µg/mL)
Patient 24231 : The images were of varying intensity of SYBR Green. In the first, it was very concentrated, and the second was less so Quantitatively, the first sample's concentration was 104.72 μg/mL, the second contained 7.16 μg/mL, and due to problems with using a cell phone, the third image was lost.
Patient 48862 : This patient was negative because there weren't any visible signs of the green light. The concentration of the first sample was lost due to an error in using a cell phone, the second sample's concentration was 1.59 μg/mL, the third sample's concentration was 1.80 μg/mL.
Errors in the observed result could be due to any of the following factors: inability to fine-tune the photographic setting of the cell phone, the inability of the cell phone to focus on the liquid drop, inability to level the drop of water with the level of the cell phone camera, improper usage of imageJ software, or varying distances used to take an adequate image within the fluroimeter.
Conclusions
Patient 24231 : Inconclusive. The first and second result are of such differing magnitude that there was an error in measurement, or at some step in the process. In addition, the third photo was lost and results were unobtainable. As a result of these two factors, it is inconclusive if the patient was positive or negative. Though, the values obtained are larger than the concentration of the positive control and lend air to this patient being positive for the disease SNP.
Patient 48862 : Negative. The two results obtained are close in magnitude. Even having lost the third image, it is likely a conclusion can be drawn from the two samples obtained. As the two values are closer to the negative control than the positive control, it is likely this patient is negative for the disease SNP.
BME 100 Fall 2015
