BME100 s2014:T Group14 L5

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Contents

OUR TEAM

Name: Caitlin R. Byrne
Name: Caitlin R. Byrne
Name: Megan McGuire
Name: Megan McGuire
Name: Wade Savage
Name: Wade Savage
Name: Theo Hall
Name: Theo Hall


LAB 5 WRITE-UP

Background Information

SYBR Green Dye

SYBR Green I is a dye that is used as a nucleic acid stain in molecular biology. The dye binds to DNA and results in a DNA and dye complex that reflects blue light and emits a green light that binds to double stranded DNA. This can be used in some methods of a quantitative polymerase chain reaction. This dye can also work on RNA, because RNA also is made up of nucleic acids. The dye will bind to the RNA and work in the exact way it would when bound to the DNA.

SYBR Green I is declared safe to work with and does not have waste disposal issues. However is has the cabable of binding to DNA with a high affinity. This means that safety gloves must be worn at all times to prent the SYBR Green dye from getting on the skin.


Single-Drop Fluorimeter

A flourimeter uses optical caustics in applied to flourescence to observe and measure the amount of flourescence. Flourescence can occur when a wavelength excites a molecule causing the emission of light. In this experiment, the SYBR Green when attached to the DNA nucleotides should glow when placed on the Flourimeter. Optical caustics causes light to be concentrated near the surface of the drop in order to increase the intensity of the flourescence. This is what can be eventually captured by our cell phones.

The flourimeter is made of generic plastic, and a drop is placed on a special type of slides with a rough surface with a micropipette. Once aligned, a blue light is shown through the drop causing the SYBR Green to flouresce and an image to be captured on a cell phone to later be recorded on the computer program ImageJ.


How the Fluorescence Technique Works

First, the flourimeter is set up by turning on the Blue LED and turning on and putting a smart phone on a cradle in front of the flourimeter. From this position, adjust the flourimeter to the height of the cell phone's camera in order to get a picture of the drop sideways and not from any other angle. This can be done by placing books or other materials underneath the flourimeter. After placing the drop of SYBR Green on the slide and adding the DNA to the dye, the dye will bind to the nucletide in the DNA. So, when the SYBR Green is exposed to the blue light, it will start to flouresce, putting a dark container over the reaction will make the flourescence easier to see in the picture taken by your cell phone.


Procedure

Smart Phone Camera Settings

  • Type of Smartphone: iPhone
    • Flash: Off
    • ISO setting: Standard on the Phone
    • White Balance: Standard
    • Exposure: Standard
    • Saturation: Standard
    • Contrast: Standard


  • Type of Smartphone: Droid Lucid
    • Flash: Off
    • ISO setting: Standard
    • White Balance: Standard
    • Exposure: Standard
    • Saturation: Standard
    • Contrast: Standard


Calibration

After setting up the stand on which we place the slide (rough side up), we made sure that the cell phone that would be taking the pictures was level with the slide. Because SYBR Green 1 dye is light sensitive, it is best to conduct the experiment by taking photos in a dark environment.  Our "dark room" in a sense is provided by a black file folder box.  This helps block out light when the picture is taken.    Keeping in mind that we were working with a light sensitive material, we kept SYBR Green underneath tin foil as to not hinder the experiment. (Personal Photo)
After setting up the stand on which we place the slide (rough side up), we made sure that the cell phone that would be taking the pictures was level with the slide. Because SYBR Green 1 dye is light sensitive, it is best to conduct the experiment by taking photos in a dark environment. Our "dark room" in a sense is provided by a black file folder box. This helps block out light when the picture is taken. Keeping in mind that we were working with a light sensitive material, we kept SYBR Green underneath tin foil as to not hinder the experiment. (Personal Photo)

The smart phone to be used in the experiment should be formatted to have the flash off at least. It's also important to have a timer on the cell phone, in order to leave it in the dark and then take the picture. It should be safe to set the timer for 5-10 seconds in order to get good picture. After setting the phone up in the camera program, put the phone in a cradle provided in front of the flourimeter and then measure the centimeters of the phone from the drop.

  • Distance between the smart phone cradle and drop = 6.3 cm


Solutions Used for Calibration [Instructions: See worksheet page 6.]

Initial Concentration of 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
1 80 80 0.5
0.25 80 80 0.125


Placing Samples onto the Fluorimeter

  1. Obtain materials from the instructor.
  2. After the set up described above, set your micropipette to 80 microliters of the SYBR Green I dye and place it in the middle of the first two ride of the slide; making sure the drop is rounded and that all of the solution is in one drop.
  3. Switch pipette tips and add 80 microliters of the DNA solution on top of the previous drop of SYBR Green I.
  4. Now, align the slide with the drop on top so that the Blue LED is shining through the sample.
  5. Now, activate the timed picture on you cell phone and cover the flourimeter with AND the camera under a light box. Or basically anything that obstructs as much stray light as possible.
  6. Take 1 image of the drop.
  7. Remove the box, making sure not to move the camera, and if you do adjust it back into position using the measurement you took before taking picture to put the camera back into place.
  8. Use the micropipette to remove the solution and dispose of it appropriately.
  9. Now repeat the steps, adjusting the slide each time for as many samples you need to take pictures of each DNA solution.
  10. After you have taken all the images, upload them onto a computer and use the ImageJ program to measure values.
  11. Then use these values to process your data.


Data Analysis

Representative Images of Samples

Positive DNA Test
The SYBR Green 1 dye causes the diseased DNA to glow when exposed to the blue light. (Personal Photo)
The SYBR Green 1 dye causes the diseased DNA to glow when exposed to the blue light. (Personal Photo)

Negative DNA Test
In the absense of diseased DNA SYBR Green 1 does not make the drop glow green, and it's a fairly obvious difference in color. (Personal Photo)
In the absense of diseased DNA SYBR Green 1 does not make the drop glow green, and it's a fairly obvious difference in color. (Personal Photo)


Image J Values for All Samples

PCR Product Table

PCR Product Tube Label

dDNA

ndDNA

1a

1b

1c

2a

2b

2c

Volume of Dilute PCR Product Solution

80

80

80

80

80

80

80

80

Volume of SYBR Green 1 in Solution

80

80

80

80

80

80

80

80

Intdens based on 3 Separate Drop Measurements

2949516

295136

414970

240148

382969

269159

837877

305779

'

2648136

164943

297687

238968

434590

278027

795755

287146

'

2942463

228896

525723

247055

427061

292295

8838335

335964

Average Intdens Value

2846705

229658.3

412793.3

242057

414873.3

279827

3490656

309629.7

PCR Product Table Calculations

PCR Product Tube Label

dDNA

ndDNA

1a

1b

1c

2a

2b

2c

Average IntDens Value

2846705

229658.3

412793.3

242057

414873.3

279827

3490656

309629.7

PCR Product Concentration

3.096757

-0.08891

0.134016

-0.07382

0.136548

-0.02784

3.880622

0.008438

Corrected PCR Product Concentration

37.16109

-1.06691

1.608195

-0.8858

1.638578

-0.33409

46.56747

0.101252

      • Just to note, we seem to have a mismeasurement on 1a measurement 2 we believe it's human error, or just taken down wrong

[Instructions: See worksheet page 8. To save time on typing a new Wiki table from scratch, use THIS TOOL to auto-generate a Wiki table: Excel-to-Wiki Converter. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code.]


Fitting a Straight Line


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 concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as threshold values for determining whether an unknown (patient) sample is truly positive or negative.

Your positive control PCR result was 37.16109 μg/mL

Your negative control PCR result was -1.06691 μg/mL

Write-in each patient ID and give both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed

Patient 40197 F : There was no florescence observed from this test drop. The intden values were all around 1.608195.

Patient 90029 M : There was no florescence observed from this test drop. The intden values were all around 0.101252.

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 40197 F : When compared to the positive and negative control value, this value seems to be slightly positive although the picture appears negative.

Patient 90029 M : When compared to the positive and negative control value, this test seems to be slightly positive as well even though the pictures appear negative.



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