BME100 s2014:W Group11 L5

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OUR TEAM

Name: Christopher Stark
Name: Kandace Donaldson
Name: Cassiana Wright
Name: Jordan Nelson
Name: Yash Jain


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR Green I is a fluorescent dye used for binding double-stranded DNA so that quantification and visualization is possible. SYBR Green is a synthetic, asymmetrical cyanine dye that was designed to work throughout a wide range of temperatures, bind in a way that does not interfere with enzymes such as DNA polymerases and nucleases, and accurately quantify DNA in varying concentrations. Real time PCR is the most common use for the dye, where the fluorescence increases as each PCR cycle amplifies the DNA so you can visually observe amplification.




Single-Drop Fluorimeter

The Single-Drop Fluorimeter is a device designed to help detect any presence of double stranded DNA in a liquid sample by measuring its fluorescence. It is essentially a small plastic device with a space on top for a multi-welled slide. The slide is illuminated by a single blue LED light which is turned on by a switch located on the right hand side of the Fluorimeter. The slide of the fluorometer is positioned so that the blue LED can iluminate any drop placed over a designated well.

Fluorimetergroup11.jpg
The front top portion of the fluorimeter is shown. Illuminated droplet can be seen.




How the Fluorescence Technique Works

The fluorescence technique works by adding a fluorescent die to a liquid with an unknown percentage of double stranded DNA. The slides used in this analysis have a hydrophobic Teflon film on one side of the slide, designed to hold each sample in place. The film contains breaks which act as wells where liquids can be held in precise quantities. These breaks also keep each of the drops apart from one another so that the samples do not mix. All these traits make it easier to observe and analyze our samples when using the LED. When the samples are placed in the LED's range, any DNA stands at the top of the drop sized sample can be seen because the light causes them to fluoresce.



Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Nokia Lumia 928
    • Flash: Off
    • ISO setting: 3200
    • White Balance: Auto
    • Exposure: 2
    • Saturation: N/A
    • Contrast: N/A


Calibration


The camera was placed in a cradle that kept it upright and facing the fluorimeter from a side angle nearly edge-on. It was kept at a distance of 13 centimeters from cradle to the drop, which was chosen to keep the drop in focus for the camera and remained constant throughout the experiment. An eraser was placed in the cradle with the phone in order to keep it snug against the side and in a consistent position. It was also necessary for the fluorimeter to be raised to a height that was suitable for the height of the camera so it was placed on top of a calculator.

67547998423.jpg

  • Distance between the smart phone cradle and drop = 13 centimeters


Solutions Used for Calibration

Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL Volume of the 2X DNA solution (μL) Volume of the SYBR Green I Dye solution (μL) Final DNA concentration in SYBR Green I solution (μg/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. Place the slide, smooth side down, in the fluorimeter (make sure it's on).
  2. Check the height of the flourimeter and camera position.
  3. Pipet 80μL drop of the SYBR Green I solution between the first two clear circles in the middle of the slide.
  4. Pipet 80μL drop of the sample solution on top of the SYBR Green I drop.
  5. Move the slide so that the fluorimeter's light illuminates the center of the drop and focuses light on the other side.
  6. Place the lightbox over the setup with the flap still open and check the camera focus again.
  7. Take picture with the camera using a set timer with the lightbox flap down.
  8. Check the picture.
  9. Pipet the drop off of the slide and into the liquid waste container.
  10. Move the slide to the next set of clear circles in preparation for the next drop.
  11. Repeat steps until all slide positions have been used and then replace the slide with a new one.


Data Analysis

Representative Images of Samples

Imagejpositive.jpg

Sample with DNA (Positive Control)

Imagejnegative.jpg

Sample with no DNA (Negative Control)


Image J Values for All Samples

Set Area Mean IntDen drop Intden/area ' ' ' ' ' ' ' ' ' ' '
5 40443 8708426 215.3259155
1 26700 3603137 134.9489513
0.25 31888 3904086 122.4311967
Area 1 2 3 Intden 1 2 3 avg intden intden/area 1 2 3 avg PCR Product concentration Corrected Concentration
control "+" 24164 33340 32796 5935034 5733849 6214941 5961274.667 245.6147161 171.9810738 189.5030187 202.3662695 4.353497315 52.24196778
control "-" 27960 28148 26952 2634292 4594063 2243889 3157414.667 94.21645207 163.2109919 83.2550089 113.5608176 -0.146398904 -1.75678685
A1 43012 30232 20364 8556840 5916286 4822294 6431806.667 198.9407607 195.6961498 236.8048517 210.4805874 4.76466113 57.17593356
A2 31908 44900 30804 6063436 7480390 5670458 6404761.333 190.0287075 166.6011136 184.0818725 180.2372312 3.232188052 38.78625662
A3 33908 38724 31868 5571646 7873538 6476711 6640631.667 164.3165625 203.3245016 203.2355655 190.2922098 3.741687856 44.90025427
B1 22064 20108 31680 1701487 1883730 3583667 2389628 77.11598078 93.68062463 113.1208018 94.63913573 -1.105186941 -13.26224329
B2 30332 22200 19016 2329264 2527096 1937352 2264570.667 76.79229856 113.8331532 101.880101 97.50185089 -0.960129167 -11.52155
B3 27608 20360 23712 3234192 2178949 4335590 3249577 117.1469139 107.0210707 182.8437078 135.6705642 0.973932818 11.68719382
Patient A 3.912845679 46.95414815
Patient B -0.36379443 -4.365533157


Fitting a Straight Line

Callibration Line.png



PCR Results Summary


Instructor's summary: You completed 8 PCR reactions in a previous lab. You used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentration of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples are used to determine the threshold values for determining whether an unknown (patient) sample is truly positive or negative.

Your positive control PCR result was 4.35 μg/mL 

Your negative control PCR result was -0.146 μg/mL


and a quantitative description (μg/mL) of what you observed

Patient 10072: When DNA from this patient was combined with the SYBR green dye and viewed in the fluorimeter, it produced an average Intdens reading of 3.91 μg/mL

Patient 83576 : When DNA from this patient was combined with the SYBR green dye and viewed in the fluorimeter, it produced an average Intdens reading of -0.364 μg/mL

Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion.

Patient 10072 : Each of the samples from this patient very closely resembled the positive control. All drops glowed bright fluorescent green in the fluorimeter. This patient appears to have tested positively for the gene of interest because the SYBR green dye was able to attach to double-stranded DNA molecules and fluoresce. Since the dye was able to do this, the PCR reactions must have produced many copies of the target strand of DNA, meaning it was present in the samples from this patient.

Patient 83576 : Each of the samples from this patient very closely resembled the negative control. None of the drops glowed or fluoresced when viewed in the fluorimeter. This patient appears to have tested negatively for the gene of interest because the SYBR green dye did not bind to any double-stranded DNA molecules to fluoresce. Since no double-stranded DNA molecules were produced in the PCR reaction, the patient did not have the target sequence of DNA.