BME100 f2016:Group1 W1030AM L5

From OpenWetWare

Jump to: navigation, search
BME 100 Fall 2016 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help
Image:BME494_Asu_logo.png

Contents

OUR TEAM

Carlye A. Frisch
Carlye A. Frisch
Kendra M. Gibble
Kendra M. Gibble
Anna Rothweil
Anna Rothweil
Gabrielle F. Wipper
Gabrielle F. Wipper
Nicholas C. Whitley
Nicholas C. Whitley


LAB 5 WRITE-UP

(pictured from left to right: Gabrielle F. Wipper, Carlye A. Frisch)


(pictured: Gabrielle F. Wipper)


PCR Reaction Report

Overall, we found that this lab was a great learning experience in PCR analysis. The pre-lab reading was very helpful. We found that it prepared us well for the lab, especially the pre-lab quizzes where we focused on each lab day's main concepts. After a few trial and errors, we had a much better understanding over the first and second stop on the pipettor; the first stop collects the solution, whereas the second stop releases the solution. The final reactions did have the same amount of liquid and some liquid was left in the tubes with the DNA samples and PCR reaction mix. We did not have to change our labeling scheme throughout this lab.

Fluorimeter Procedure

Imaging set-up

First, the fluorimeter was set up. A hydrophobic slide was placed onto the device with its smooth side down. The blue LED light was turned on to lighten the first two rows of the slide. A cradle with a smartphone on it was set to take pictures of the slide. The distance between the camera of the smartphone and the first two rows of the slide was adjusted in a way that the camera was focused at that part of the slide and as close to it as possible. It was also important to higher the fluorimeter (in our experiment, we used plastic trays to do so) so that the camera would take pictures of the drops on the slide sideways.


Placing Samples onto the Fluorimeter

  1. Step 1: First, you put a 160 microliter drop of water onto the slide, ensuring that it is in the middle of the first two rows. The drop should look similar to a marble.
  2. Step 2: Next, switch the blue LED light on.
  3. Step 3: Ensuring that the flash is off on your smartphone, turn on the camera and set the phone at a 90 degree angle from the slide.
  4. Step 4: Adjust the smartphone so it is at least 4 cm away from the slide. Record the distance. The image on the camera cannot be blurry.
  5. Step 5: Place an 80 microliter drop of SYBR Green I in the middle of the first two rows of the slide. Next, add another 80 microliter drop of calf thymus to the first drop. This is considered a "sample."
  6. Step 6: Move the slide so the blue LED is focused on the middle of the drop.
  7. Step 7: Set the timer on the camera to 3 seconds so that a picture can be taken after covering the fluorimeter and camera with the light box.
  8. Step 8: Take three images of the sample, ensuring that the camera is focused.
  9. Step 9: Remove the box without removing the smartphone.
  10. Step 10: Use pipette to remove the drop from the surface. Move the slide to the next position.
  11. Step 11: Repeat steps 5 through 10 for the other concentrations of calf thymus DNA.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High (concentration= 5.0):

Low (concentration= 0.5):

Zero (concentration= 0):


Calibration Mean Values

Initial Concentration of 2X Calf Thymus DNA Solution (μg/mL) Final DNA concentration in SYBR Green I Solution (μg/mL) Sample Number RAWINTDEN Drop-Background Image 1 RAWINTDEN Drop-Background Image 2 RAWINTDEN Drop-Background Image 3 Mean Standard Deviation
5 2.5 C-1 1484689 1756158 1753590 1664812.33 155996.67
2 1 C-2 5628689 5621361 5617872 5622640.67 5520.87
1 .5 C-3 5188687 5184025 5191233 5187981.67 3655.40
0.5 .25 C-4 3752754 3764623 3759634 3759003.67 5959.553703
0.25 .125 C-5 5854528 5860678 5875705 5863637.00 10894.18
0 0 C-6 96988 102741 89040 96256.33 6879.74


Calibration curves
Image:excelg1lab5.1.jpg Image:excelg1lab5.2.jpg

Note: Error bars of the standard deviations have been added to both plots, but are invisibly small.


Images of Our PCR Positive and Negative Controls

Positive Control:

Negative Control:


PCR Results: PCR concentrations solved

PCR Product Tube Label MEAN (of RAWINTDEN DROP-BACKGROUND) PCR Product Concentration (μg/mL) Step 5 Calculation Total Dilution Initial PCR Product Concentration (μg/mL) (Step 6 Calculation)
G1- 474035.333 -1.262982334 12 -15.155788
G1+ 6661927 1.8309635 12 21.971562
G1 1-1 682278.666 -1.158860667 12 -13.906328
G1 1-2 1144565.33 -0.927717335 12 -11.13260802
G1 1-3 477864.666 -1.261067667 12 -15.132812
G1 2-1 1407615 -.07961925 12 -9.55431
G1 2-2 925541 -1.0372295 12 -12.446754
G1 2-3 929748.333 -1.035125834 12 -12.42151



PCR Results: Summary

  • Our positive control PCR result was 21.971562 μg/mL
  • Our negative control PCR result was -15.155788 μg/mL


Observed results

  • Patient 49774:

The first test for this patient did not show much green in the drop of solution. The second and third test look very similar to this first one as the drop appears to be clear. These tests look similar to the negative control. In the first test for this patient, the PCR product concentration was -1.158860667 μg/mL while the initial concentration was -13.906328 μg/mL. In the second test the PCR product concentration was -.927717335 μg/mL and the initial concentration was -11.13260802 μg/mL. In the third test, the PCR product concentration was -1.261067667 μg/mL and the initial PCR product concentration was -15.132812 μg/mL. The first test's mean we found to be 682278.666, the second's was 1144565.33, and the last one's was 477864.666.

  • Patient 38123:

The first, second, and third test appear to be clear solution from the pictures taken. They all look similar in comparison to the negative control solution. In the first test, the PCR product concentration was -.7961925 μg/mL while its initial PRC product concentration was -9.55431 μg/mL. The second test's PCR product concentration was -1.0372295 μg/mL and its initial PCR product concentration was -12.446754 μg/mL. In the third test the PCR product concentration was -1.035125834 μg/mL while the initial PCR product concentration was -12.42151 μg/mL. The first's mean was 1407615, the second's was 925541, and the last one's was 929748.333.

Conclusions

  • Patient 49774:

The three test results of the above patient are really similar, the fact which shows the reliability of the test done. The three values are between -11.13 and -15.14, resembling the initial PCR concentration of the negative control (-15.16). However, neither value exceeds the latter value, thus it can't be stated that the patient is truly negative. The test results are nothing like the positive control. The final conclusion made for patient 49774 was negative (as of 11/1/2016). Nevertheless, having further analyzed our data since then, inconclusive would be the accurate judgment.

  • Patient 38123:

The relatively small deviation of the results of the three tests demonstrates that the test done was reliable. The values examined are very similar to the results of the samples from patient 49774. In this case neither can it be defined if patient 38123 is truly negative for the test even though the tendency of initial PCR concentrations is surely negative and not positive. Although the final conclusion made previously was negative, the outcome is rather inconclusive in case the concentration of the negative control is considered a valid threshold value.


Personal tools