To set up the fluorimeter, the excitation light needs to be turned on, which is the blue switch for the Blue LED light.
Then the camera on the smartphone needs to be turned on.
After the settings are adjusted on the smartphone, it is placed in the cradle at a right angle because it makes filtering easier in ImageJ. The height is also adjusted so that the camera can take a picture of the drop from the side.
The distance is then adjusted between the smartphone and the cradle so that it is 4cm or more away from the slide so that the drop does not appear blurry.
The distance between the drop and smartphone in the cradle is recorded by a ruler and should not be moved from its position because the light collected can change the image that is captured.
Distance between the smart phone cradle and drop = 5 cm
Solutions Used for Calibration
Placing Samples onto the Fluorimeter
Place a 80* microliter drop of SYBR GREEN I in the middle of the first two rows with the pipettor. Then add 80* microliters of one solutions that is in the table above, which will make a sample
Move the slide so that the blue LED light is focused on the side of the drop.
Set the timer to take a picture after the light box is shut. The light box removes most of the stray light, so it is okay if a little gets in.
Make sure the drop is in focus and take three pictures of the drop.
Be cautious when removing the box, so that the distance between the smartphone is not tampered with. If the smartphone needs to be adjusted, use a ruler so that the exact position it was at can be returned to.
Remove the 160 microliter drop from the slide with the pipettor and move the slide to the next two rows.
Repeat steps 1-5 for the other concentrations that are listed in the table above.
Several of the slides can be used, since they are inexpensive, if the calibration needs to be rerun or if a mistake happens.
Data Analysis
Representative Images of Negative and Positive Samples Above is a photo of the PCR TUBE Labeled Negative droplet on the Fluorimeter
Above is a photo of the PCR TUBE Labeled Positive droplet on the Fluorimeter
Image J Values for All Calibrator Samples
Calibration curve
PCR Results Summary
Our positive control PCR result was 2.76 μg/mL
Our negative control PCR result was 0.4301 μg/mL
Observed results
Patient 31366 : 6.89 μg/mL; When the SYBR Green was added to the drop, the droplet reflected a green light
Patient 26606 : -3.23 μg/mL; When the SYBR Green was added to the drop, the droplet did not reflect a green light
Conclusions
Patient 31366 : This patient was positive because when the SYBR Green was added to the drop, it reflected a green light similar to that of the positive control. This patient has the Calf Thymus gene, therefore the light expressed a green reflection.
Patient 26606 : This patient was negative because when the SYBR Green was added to the drop, it did not reflect a green light, similar to that of the negative control. Different from Patient 31366, since this patient did not have the Calf Thymus gene, no green light was reflected.
SNP Information & Primer Design
Background: About the Disease SNP
Building block of DNA
A variation that is in DNA
The latin name is Homo Sapiens
21:34370656
It is pathogenic
KCNW2
Long Qt syndrome
Potassium voltage-gated channel
An alternative form of a gene that may cause mutation
BME 100 Fall 2014
