# BME100 f2014:Group32 L5

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# OUR TEAM

 Name: Isaiah Gonzales Name: Fahhad Ashour Name: Blake Bosold Name: Royal Boggs Name: Neal Viswanath Name: Nava Nozari

# LAB 5 WRITE-UP

## Procedure

Smart Phone Camera Settings

• Type of Smartphone: iphone
• Flash: no flash
• ISO setting: 800
• White Balance: Auto
• Exposure: 2
• Saturation:326 ppi.
• Contrast: 800:1

Calibration
Instructions:

1. Using the Blue LED switch, turn on the excitation light.
2. With your smart phone turn on the camera and check the settings. If the settings can be adjusted, do so accordingly as described above.
3. Place the smart phone in the cradle at a right angle from the slide. If necessary, adjust the height of the fluorimeter using the plastic trays. The camera should take a picture of the drop sideways as to make filtering the blue light easier using ImageJ.
4. Without making the image blurry, adjust the distance between the smartphone/cradle and the first two rows of the slide so that it as close as possible.
5. Using the ruler provided in lab, measure the distance between the smart phone cradle and drop. Be careful not to move any equipment when measuring the distance as this could result in a significant difference from one image to the next.
• Distance between the smart phone cradle and drop = 7 cm

Solutions Used for Calibration

 Initial concentration of Calf Thymus DNA Solution (ug/mL) Volume of the DNA solution (uL) Volume of the SYBR Green I Solution (uL) Final DNA in the SYBR Green Solution (ug/mL) 5 80 80 2.5 2 80 80 1 1 80 80 0.5 0.5 80 80 0.25 0.25 80 80 0.125 0 80 80 0

Placing Samples onto the Fluorimeter

1. Using the pipettor, place a 80 microliter drop of SYBR GREEN I in the middle of the first two rows of the slide. Then add 80 microliters of one of the calf thymus (water blank) solutions listed in the table above. This is now considered a "drop" or "sample."
2. Move the slide so that the blue LED light is focused by the drop to the middle of the black fiber optic fitting on the other side of the drop.
3. Adjust the timer on the camera to take a picture after covering the fluorimeter and camera with the light box. The light box should remove as much stray light as possible.
4. Take three pictures of the drop.
5. Remove the box. Do not move the smartphone. To adjust for movement, use the ruler to return to the previous distance.
6. Remove the 160 microliter drop from the surface using the pipettor and move the slide to the next position.
7. Repeat steps 1-6 for the other concentrations of calf thymus DNA.
8. If mistakes are made, rerun the calibration.

Fluorimeter Set Up Image

## Data Analysis

Representative Images of Negative and Positive Samples

Image J Values for All Calibrator Samples

Calibration curve

PCR Results Summary

• Our positive control PCR result was 38.11931047 μg/mL
• Our negative control PCR result was 25.04827766 μg/mL

Observed results

• Patient 39917 : The images for this patient did not glow a bright green and were usually dark blue or light blue as is reflected by the light. There was a shadowy air bubble for the PCR reaction solution that was perhaps trapped inside of the drop. The solution values for the three different images that were analyzed were: 19.59540867, 21.48631432, 24.92983236 μg/mL respectively.
• Patient 34918 : The images for this patient as well did not glow green and were just a clear color with the blue led light reflected onto it. There was a bubble of the sample in the center as well. The solution values for these three different images were: 27.50419844, 19.51289755, 23.46186438 μg/mL respectively.

Conclusions

• Patient 39917 : Because these images did not glow and the values calculated for the initial PCR solutions product concentrations was directly in line with the negative sample initial PCR product concentration, these samples were negative as well.
• Patient 34918 : This sample is negative by the same reasoning: the images did not glow a bright green indicating that there was a reaction happening in solution and the values calculated for the initial PCR solution product concentrations was inline with the negative sample initial PCR product concentrations.