BME100 f2016:Group1 W8AM L5

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OUR TEAM

Name: Audriana Sedmak
Name: Benjamin Robles
Name: Emma Rodriguez
Name: Mitchell Miranda
Name: Spencer Brimley


LAB 5 WRITE-UP

PCR Reaction Report

To start the experiment samples of DNA from two separate patients was provided, as well as a sample that was known as positive for PCR gene and one that was known to be negative for the PCR gene, and PRC reaction mix. A micropipette was used to transfer 50 uL of each sample of DNA from the patients as well as the positive and negative DNA samples into knew PCR reaction tubes. Each sample was put into a different tube, and a new pipette tip was used for each sample. 50 uL of the PCR reaction mix was then placed into each different tube, with a new pipette tip was used each time again to keep the samples pure.Visually it appeared as though all of the tubes had the same volume after the DNA samples and the PCR reaction mix had been added. The new reaction mixes were placed into the PCR machine which ran until the reaction was run to completion. When using a micropipette it is important to change the tip every time you work with a new sample and to set the desired volume on the pipette. The first stop verses the second stop of the pipette insures that the solution is not let out too quickly and also ensures that all of the solution is gotten out of the pipette tip.

Fluorimeter Procedure


Imaging set-up

  1. Set up the light box so the front is open.
  2. Put the fluorimeter in the back of the box on a clean, flat surface.
  3. Position the camera so that it is able to "see" the top of the sample slides and view the drops horizontally.
  4. Insert slide facing smooth-side down.


Placing Samples onto the Fluorimeter

  1. Place 80 µl of SYBR green between the first two rows of the slide.
  2. Add 80 µl of the DNA sample to the SYBR green.
  3. Using a timer, take a picture of the sample on the fluorimeter inside the light box.
  4. Remove the 160 µl sample and move the slide to the next row in preparation for the next sample.
  5. Repeat steps 3 thru 6 for all the samples.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA
5 μg/mL Sample
5concen.jpg

0.5 μg/mL Sample
1concen.jpg

0 μg/mL Sample
0concen.png


Calibrator Mean Values

DNA Concentration (μg/mL) Mean Values Standard Deviation
2.5 176503 1688.562406
1 172361.3333 3843.387351
0.5 168355 413.6822452
0.25 132315.6667 4877.734549
0.125 114951 6457.839964
0 10741.66667 5409.972304


Calibration curves
Calibrationcurve.png PCR Results.png

Images of Our PCR Negative and Positive Controls

Positive Control Sample
Poscontrol.png

Negative Control Sample
Negcontrol.png


PCR Results: PCR concentrations solved

Sample Mean Values Standard Deviation
Positive 2484906.667 16912.17199
Negative 414204 12988.15811
2- 1 447098.3333 11840.88022
2- 2 510286.3333 19808.63186
2- 3 498401.6667 10001.80465
3- 1 2464185.667 42453.08399
3- 2 2275727.667 40950.64978
3- 3 2407322.667 52543.00891


PCR Results: Summary

  • Our positive control PCR result was 1 μg/mL
  • Our negative control PCR result was 0 μg/mL


Observed results

  • Patient 28313: 0
  • Patient 36189: 1


Conclusions

  • Patient 28313: Negative, these values were very close to the negative control group.
  • Patient 36189: Positive, these values were much higher than the first, and were significantly related to the positive control group.