BME100 s2016:Group2 W1030AM L5

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OUR TEAM

Name: Bailey Gasvoda
Name: Erik Drager
Name: Ishitha Jagadish
Name: Earl Brown
Name: Destinee Martin-Karim
Name: Logan Luke

LAB 5 WRITE-UP

PCR Reaction Report

Was the Experiment Successful?

All lab members had prior experience with micropipettors, and therefore there was no problem with transferring the proper solutions to the corresponding labeled tubes. The prepared tubes had the same amount of liquid in the end, meaning that the correct amounts of liquids were transferred properly into the right containers. These containers were labeled according to the mixtures that needed to be prepared; however, these labels did not have to be changed since the required liquids were transferred successfully.


Using a Micropipettor

In order to acquire a sample using the micropipettor device, the user had to set the sample amount (which is in microliters), press the first stop, place the micropipette just below the substance meniscus, and release the stop slowly. In order to release the micropipette sample, the user had to push all the way to the second stop slowly then release the pipette tip into a waste container. After each time the micropipettor was used, the old pipette tip was discarded into a waste container. Then, a new pipette tip was inserted onto the micropipettor.


Steps of Running PCR Reactions on Samples of DNA (Lab C)

  1. Two sets of four tubes of patients' DNA, one set per patient, were assigned for analysis. Two additional tubes, positive and negative controls, were used in order to confirm that the results were accurate. These tubes were kept in a rack for easy access.
  2. A Sharpie permanent marker was used to label the empty tubes on their sides. Each set of four tubes of patients' DNA were labeled with the respective patients' IDs.
  3. 50 μL of PCR reaction mix was transferred into tube labeled as the positive control.
  4. In order to avoid cross-contamination, the appropriate micropipetting techniques were used properly. A new pipette tip was placed on the micropipette, and then 50 μL of the mix of the positive control DNA and primer was dropped into the tube labeled as the positive control.
  5. The previous two steps were repeated several more times for: the negative control, the four tubes of the first patient's DNA, and the four tubes of the second patient's DNA. Note: There was a corresponding DNA/ primer mix for each tube.
  6. The final tubes had approximately the same amount of fluids (100 μL total), their differences are virtually negligible.
  7. Close the lids tightly on your PCR reaction tubes.
  8. The lids of the total of ten tubes were closed tightly, and then the ten tubes were placed into the heating block of the OpenPCRMachine.
  9. Then, proper heating and cooling cycles were applied in order to have a successful PCR reaction. These steps were described in a previous lab report.


Preparation of PCR Products to Capture Images (Lab D)

Sample Tubes Used

  • 8 tubes containing 500 microliters of buffer solutions (these tubes were marked with red dots on the lids)
  • 2 tubes containing 1,000 microliters of SYBR GREEN 1 Solution (marked with an "S" on the lids)
  • 1 tube containing 1,000 microliters of pH 8 water (marked H2O)
  • 8 tubes containing the PCR reaction samples which were assigned and underwent the PCR process in Lab C

Steps (Lab D)

  1. Each tube containing buffer was labeled corresponding to the labels of the PCR reaction samples.
  2. The micropipettor was set to 120 microliters and a new, clean disposable pipette tip was attached.
  3. The entire 100 microliters of the liquid from each of the eight PCR tubes were transferred into the corresponding eight buffer tubes (according to the labels).
  4. The lids of the buffer tubes were closed, and these tubes were lightly tapped in order to properly mix the buffer with the added DNA.


The Use of the Fluorimeter to Take Measurements of the PCR Samples

The same procedure that was used to set up the phone camera and the fluorimeter for calibration with the calf thymus DNA solutions was used for the PCR samples.

Fluorimeter Procedure

Phone camera set-up

Using the stand that was provided with the light box, an iPhone 6 was placed on the stand; therefore, the camera of a phone was used. The camera was positioned at a 90 degree angle from the slide such that it captured images of drops that looked like oblate spheroids. Flash on the iPhone 6 camera was deactivated, and no other settings were altered. ImageJ was downloaded onto a macbook and was used to analyze the images that were taken. These images were sent from the iPhone 6 to the macbook that had ImageJ installed.


Placing Samples onto the Fluorimeter

160 microliters of water

  1. 160 microliters of water was placed on the slide in the fluorimeter using a micropipettor so that the water formed an oblate spheroid (oval sphere) shape.
  2. The blue LED light was turned on using the light switch.
  3. The iPhone 6 camera was set-up to take images, as was described above.
  4. The camera was placed at a 90 degree angle from the slide, making sure that the camera took a picture of the entire drop from a sideways point of view. The height of the stand was adjusted by putting plastic trays underneath the stand.
  5. The distance between the camera and the cradle was adjusted so that the image was not blurry in order to obtain acceptable readings. This distance should have been at least 4 cm apart, which was measured with a ruler, and then recorded.
  6. A picture of the oblate spheroid of water was taken to ensure that the phone settings were working well.
  7. The 160 microliters of water was then removed from the slide to prepare for the next part of the experiment.


Calf Thymus DNA Samples

  1. 80 microliters of the SYBR GREEN was placed on the slide using a micropipettor so that it formed an oblate spheroid (oval sphere) shape.
  2. Then, 80 microliters of one of the calf thymus solutions was added to this drop.
  3. The drop was aligned so that the blue light was focused on it onto the black fiberoptic fitting on the other side.
  4. Using the timer on the camera, a picture of the drop was taken when the light box was closed. Light was eliminated from entering the light box as much as possible.
  5. Three focused images of this drop was taken.
  6. Remove the box and carefully remove the 160 microliter drop.
  7. These steps were repeated for the two other concentrations of calf thymus DNA. The concentrations that were used were high, low, and zero.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

  • High Concentration

Calibrationpichigh.png

  • Low Concentration

Calibrationpiclow.png

  • Zero Calf Thymus DNA

Calibrationpiczero.png

Calibrator Mean Values


Calibrationtablefixed.png


Calibration curves

Group2dotplot1fixed.png Group2dotplot2.png

Images of Our PCR Negative and Positive Controls

  • Negative Control

Negativecontrolpic.png

  • Positive Control

Positivecontrolpic.png

PCR Results: PCR concentrations solved

Pcrsolutionstablefixed.png


PCR Results: Summary

  • Our positive control PCR result was 16.308 μg/mL
  • Our negative control PCR result was 1.439 μg/mL


Observed results

  • Patient 67115: The three samples from this patient appeared colorless. They were not bright green like the positive control sample. The calculated PCR concentrations ranged from 0.556-1.959 μg/mL.
  • Patient 82576: The three samples from this patient resembled the negative control. They were colorless as opposed to green. The initial PCR concentrations were between 1.359-3.241 μg/mL.


Conclusions

  • Patient 67115: This patient is negative. The positive control threshold is 16.308 μg/mL while this patient was only at an average of 1.319 μg/mL for the three samples. This was almost equivalent to the negative control.
  • Patient 82576: This patient's average PCR value, 2.414 μg/mL, is similar to the negative control value. This is well below the threshold of the positive control.