BME100 f2017:Group11 W0800 L5

Our Team

Name: Braeden Malotky

Name: Mauro Robles

Name: Alexa Ng

Name: Keiko Ochoa

Name: Connor Leicken

PCR Reactions

Two patients have each submitted three replicate DNA samples to be tested for a disease marker. The six samples, in addition to one positive and one negative control, were analyzed. To test for the disease marker, each sample of DNA was mixed with a PCR reaction mix and primers, which target the specific piece of DNA associated with the disease. The eight samples were then placed in the thermocycler to amplify the DNA. The positive control and any of the samples of the patients with the disease should have high frequency of DNA because the primer was able to find and attach to the disease DNA section, thus amplifying it. There should be a low amount of DNA amplified in the negative control and the patients' samples without the disease, because the primer was unable to attach to the disease-associated DNA piece and amplify it.

Fluorimeter Procedure

Camera Set-Up

Model: iPhone 7

1. Change settings on camera

• Turn off flash
• Set ISO to 800
• Set Exposure to highest setting (2)
• Set Saturation to highest setting (if possible)
• Set Contrast to lowest setting (if possible)

2. Set camera timer to three seconds

3. Set the camera in the cradle so that the drop on the fluorimeter slide is in focus and that the camera is as close to the drop as possible

4. Measure the distance between the fluorimeter and the camera. Maintain constant distance for every photo. (Ours was 4cm)

5. Cover the entire system (fluorimeter and camera) with the box so the picture is taken in relative darkness

Using the Fluorimeter

1. Turn on the fluorimeter so that the blue light is on

2. Place the slide into the fluorimeter smooth side down.

3. Place a 80 microliter drop of SYBR GREEN I on the first two holes in the middle rows of the slide using the micropipettor. Using a new micropipettor tip, then add 80 microliters of either the calf thymus (or water blank) solutions.

4. Take three pictures of the drop using the smartphone camera procedure described above. Make sure to document the images correctly afterwards.

5. Remove the drops from the slide using the micropipettor. Move the slide forward so the next two holes are at the front and repeat steps 2-5 for the next DNA sample.

Data Analysis

Table 1: PCR Raw Data

Sample Number	Image Number	Final DNA Concentration	Area	Mean Pixel Value	RawIntDen of the Drop	RawIntDen of the Background	Minimun	Maximum	Color
1	1	0.125	46504	135.441	6298558	175878457	25	255	None
1	2	0.125	42528	127.022	5401981	165393967	37	255	None
1	3	0.125	47044	143.201	6736742	197932480	48	255	None
2	1	0.25	69476	131.232	8828402	1890958259	8	255	Green
2	2	0.25	65444	143.902	9417545	181485250	32	255	Green
2	3	0.25	62180	139.062	8645893	186035957	11	255	Green
3	1	0.5	73968	158.401	11716623	219467452	27	255	None
3	2	0.5	80252	158.168	12693271	200502424	43	255	None
3	3	0.5	80185	150.751	12087944	199536625	33	255	None
4	1	1	105792	128.667	13611992	134825536	15	255	None
4	2	1	116248	125.272	14562663	126905865	15	255	None
4	3	1	114692	128.927	14786851	128998070	14	255	None
5	1	2.5	108572	119.377	12960969	147753261	27	255	None
5	2	2.5	103184	116.355	12005943	141339941	19	255	None
5	3	2.5	107328	117.294	12588935	140756949	20	255	None
6	1	0	80596	154.013	12729926	109203822	39	255	Green
6	2	0	123208	151.694	18689945	128893087	30	255	Green
6	3	0	73032	154.514	11284471	99827747	48	255	Green
7	1	0	137412	86.805	11928032	166099184	18	255	None
7	2	0	135032	87.46	11809964	158043551	19	255	None
7	3	0	135284	90.478	12240209	167824147	10	255	None
8	1	0	82360	142.918	11823742	177234556	17	255	None
8	2	0	86848	148.175	12868732	176300973	20	255	None
8	3	0	71268	148.344	10572213	17866576	55	255	None

Pictures of Thymus DNA at 0,0.25, and 1.0