BME100 f2015:Group5 8amL5

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Owwnotebook icon.png BME 100 Fall 2015 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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OUR TEAM

Name: Sultan Alanazi
Role: Analyst
Name: Tyler Mitchell
Role: Researcher/Editor
Name: Logan Martin
Role: Researcher/Editor
Name: Maryam Alsuwailem
Role: Researcher
Name: Amber Sogge
Role: Analyst
Name: Austin James
Role: Editor

LAB 5 WRITE-UP

PCR Reaction Report

The PCR reaction went as according to plan outlined in the previous lab. The pipetting technique was performed correctly. All of the tubes were labeled as specified and the pipet tips were disposed after every use. The pre-lab reading helped us because it was the only reference we had as a team. The difference between the first and second step of the micropipettor was fully understood after actual use of the pipettor. No noticeable errors were made.

Fluorimeter Procedure

Smart Phone Camera Settings

  • Type of Smartphone: Windows Phone
    • Flash:off
    • ISO setting:1600
    • White Balance:auto
    • Exposure:Auto/High
    • Saturation:High
    • Contrast:Low


Camera set-up

  • Distance between the smart phone cradle and drop = 8 cm


Placing Samples onto the Fluorimeter

  1. Begin by wearing proper lab attire. Proper lab attire includes a lab coat, splash proof lab goggles, and nitrile gloves.
  2. Turn on the fluorimeter.
  3. Insert a slide into the fluorimeter.
  4. Set up a phone to be on camera mode and place the camera on the cradle 8 cm (or what works best) from the fluorimeter. Record the position for reference.
  5. Turn on the blue light to the fluorimeter.
  6. With a micropipette, place 80 microliters of SYBR Green I on the slide.
  7. Pipette 80 microliters of the calf thymus sample solution on top of the SYBR Green I.
  8. Make sure the blue light from the fluorimeter is shining through the sample solution mixture.
  9. Carefully, take 3 pictures of the mixture on the slide.
  10. Repeat the same process for the other solutions.



Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High Calf Thymus DNA: 5 μg/mL sample.


Low Calf Thymus DNA: 0.5 μg/mL sample.


Zero Calf Thymus DNA: zero DNA.


Calibrator Mean Values

Initial Concentration of 2x calf thymus DNA solution (micro-grams/mL) DNA Concentration in SYBR Green I Solution (ug/mL) Sample Number Rewritten Drop-background Image 1 Rewritten Drop-background Image 2 Rewritten Drop-background Image 3 Mean Standard Deviation
5 2.5 c-1 2569281 1847555 2017169 2208418 510337.349
2 1 c-2 3180329 1215525 1499105 1964986 1062025.31
1 0.5 c-3 1014690 1953874 753997 1240854 631101.186
0.5 0.25 c-4 387213 855802 259878 500964 313824.597
0.25 0.125 c-5 224730 90588 418304 244541 164753.728
0 0 c-6 111477 69280 48586 76447.7 32052.3175


Calibration curves
Dot Plot 1.

Dot Plot 2.


Images of Our PCR Negative and Positive Controls

Negative Control: Negative Control PCR Sample.

Positive Control: Positive Control PCR Sample.


PCR Results: PCR concentrations solved

PCR Product TUBE LABEL RAWINTDEN DROP - BACKGROUND (Image 1) RAWINTDEN DROP - BACKGROUND (Image 2) RAWINTDEN DROP - BACKGROUND (Image 3) MEAN Standard Deviation
G5 + 1407873 1173470 3531548 2037630.333 1299068.392
G5 - 463209 108104 204503 258605.3333 183630.5724
G5 1-1 1849175 1755004 2071948 1892042.333 162762.3451
G5 1-2 1605598 3856801 2040142 2500847 1194221.738
G5 1-3 1575054 1844147 2688212 2035804.333 580800.8121
G5 2-1 1855408 1053224 400833 1103155 728571.847
G5 2-2 265328 546038 354711 388692.3333 143407.0231
G5 2-3 141640 282421 151399 191820 78614.34615
MEAN (of RAWINTDEN DROP - BACKGROUND) "PCR Product Concentration (µg /mL)(Step 5 calculation) Total Dilution Initial PCR Product Concentration(µg /mL)(Step 6 calculation)
2037630.33 2.001291162 12 24.01549395
258605.333 0.194514069 12 2.334168829
1892042.33 1.853432046 12 22.24118455
2500847 2.471733832 12 29.66080598
2035804.33 1.999436677 12 23.99324013
1103155 1.052238382 12 12.62686058
388692.333 0.326630376 12 3.919564507
191820 0.126686911 12 1.520242931


PCR Results: Summary

  • Our positive control PCR result was 24.06 μg/mL
  • Our negative control PCR result was 2.334 μg/mL


Observed results

  • Patient 16274 : The results for patient 1 appeared to be positive. There was visible green in the drop. The values for the first patient were 22.24118455μg/mL, 29.66080598μg/mL, and 23.99324013μg/mL. The average value was 25.29841022μg/mL. The results are close to the positive control of 24.06 μg/mL.
  • Patient 42480 : The results for patient 2 also appeared to be negative. There was no visible green in the drop. The values for the second patient were 12.62686058μg/mL, 3.919564507μg/mL, and 1.520242931μg/mL. The average value was 6.022222673μg/mL. Although one reading was a little higher than normal, the overall average is closer to the negative control of 2.334 μg/mL.


Conclusions

  • Patient 16274 :This patient's results turned out positive after compared with the positive and negative control. The average value for patient 16274 was close to the positive control. Through observational analysis, the drop was green thus further confirming a positive reading.
  • Patient 42480 :The results for this patient were negative as well. The average value for patient 16274 was close to the negative control. Through observational analysis, the drop was not green thus further confirming a negative reading.