Name: Emigdio Ruiz Esquivel
LAB 5 WRITE-UP
Smart Phone Camera Settings
- Type of Smartphone:iPhone 5 S
- Flash: N/A
- ISO setting: Automatic
- White Balance: N/A
- Exposure: N/A
- Saturation: N/A
- Contrast: N/A
- 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.
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
PCR Results Summary
- Our positive control PCR result was 2.76 μg/mL
- Our negative control PCR result was 0.4301 μg/mL
- 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
- 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
- It is pathogenic
- Long Qt syndrome
- Potassium voltage-gated channel
- An alternative form of a gene that may cause mutation
Primer Design and Testing