BME100 f2016:Group11 W8AM L5

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OUR TEAM

Name: Erik Ireland
Name: Isaac Alemu
Name: Elmer Correa
Name: Maria Predtechenskaya
Name: Zachary Mizera


LAB 5 WRITE-UP

PCR Reaction Report

In the beginning, we had a set of micropipette tips along with a micropipette. We had PCR samples to mix with the lab patients data and a multitude of tubes. The method into labeling the test tube with PCR was based on two different groups. We cut the eight tubes given into two sets of four tubes so that they could fit into the cycler. Putting the group number first(G11), we had a positive and negative control tube, three tubes with DNA samples of the corresponding patient. There was no change needed in labeling, most of the labeling was just a conscious understanding of our team about what the labels meant for each test tube.


Before doing anything, the students had to put on lab coats and goggles.

After the tubes were labeled, the next step was combining the necessary elements of the PCR, such as the DNA samples from each of the patients, the PCR mix with the enzymes and Taq plymerase, and the positives and negatives. Of the PCR mix, in every labeled tube 50 microliters were added. Then, into the positive and negative tubes, 50 microliters of the positive and negative samples were added, respectively. In the tubes of DNA of the patients, 50 microliters of each patent's sample was inserted. This way, in tubes 1.1, 1.2, and 1.3, the DNA of patient 56439 was placed, and in tubes 2.1, 2.2, and 2.3, the DNA of patient 58174 was inserted.

For each addition of liquid the following steps were followed:

First, the pipette was set to the desired amount. A tube with liquid that will be transferred was opened. Next, a pipette tip was attached to pipette by firmly pressing on it. Before placing the pipette tip into the liquid, the button was pressed to the first stop. The tip was then inserted and the stopper was slowly released to suck the amount of liquid needed. Next, the tip was inserted into the new tube (with or without liquid, depending on the sequence of the addition of the liquids), and the stopper was pressed to the second stop to get out the exact amount of liquid. Finally, the tip was ejected into the cup. After everything was added to the tube, the tube lid was closed.

After filling all the tubes with the necessary fluids, they were taken to the thermal cycler which was programmed to the specific cycling program with specific temperatures and times. The lid was closed, and the cycler started working. After about one hour, the tubes were placed in a freezer for the next lab.

The pre-lab reading did help, especially with understanding proper micro-pipetting techniques and the concepts behind thermal cycling as well as knowing which mixes were needed for the final samples in the thermal cycler. This also means we had a solid understanding of the difference between the first and second stop. The first was used to collect a correct volume of liquid while going down to the second stop would have a miscalibrated suction power, collecting an incorrect volume of liquid. Pushing down to the second stop is only appropriate when dispensing collected liquid. The final liquids unfortunately did vary slightly in volume as visibly viewed at the side of the tubes. On one particular PCR reaction mix tube, there was significantly more left than in the other 7 tubes, indicating a break from proper micro-pipetting technique when it was being collected.

Fluorimeter Procedure

Imaging set-up
First, the smartphone from one of our lab group members was adjusted so that: there was no flash, the ISO was higher than 800, the exposure and saturation were as high as possible, the contrast was set to the lowest possible, and the white balance was auto. Then we placed the camera in a holder and moved the holder 9 cm away from the place where the drop was going to be. We placed the slide holder (9 cm away from phone holder). Then we inserted a slide into the slide holder. Finally we placed the black box over the whole structure and then took pictures once the liquid drop was on the slide and the light was turned on and shining through the water bead.


Placing Samples onto the Fluorimeter

  1. Place the slide onto the slide tray so that the white space is facing towards us.
  2. Then, with a micropipette add the right amount of water or mixture as a bead of liquid between the first and second rows of dots and right in the middle of the slide.
  3. Put the black box over the whole structure.
  4. Turn on the light switch.
  5. Make sure the blue light goes through the middle of the drop.
  6. Take 3 pictures of this.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA HIGH.PNG (green)111.png MEDIUM.PNG (green)111.png LOW.PNG (green)111.png


Calibrator Mean Values


Excel.PNG


Calibration curves
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Images of Our PCR Negative and Positive Controls

Positive Control

POS111.png

Negative Control

NEG111.png

PCR Results: PCR concentrations solved

PCR Results Table11.PNG


PCR Results: Summary

  • Our positive control PCR result was _459.1___ μg/mL
  • Our negative control PCR result was _461.8___ μg/mL


Observed results

  • Patient 56439 : This patient had the only observed florescence out of all the drops tested. Other than that single trial no florescence occurred with the drops being clear with a backdrop of blue from the light shined through them. However, the concentrations found were 456.5, 463.6, and 462.4 μg/mL, 2 out of three of which are closer to the negative control. This contradiction must have somehow occurred due to some error during experimentation.
  • Patient 58174 : No florescence was observed in any of the drops, with no hint of green shining through. 2 out of the 3 trials (460.3, 459.8, and 459.7) had concentrations closer to the positive control, despite the absence of florescence in the drops.


Conclusions

  • Patient 56439 : The conclusion made for this patient is negative due to the quantitative analysis showing 2 out of the 3 trials having calculated concentrations closer to the negative control.
  • Patient 58174 : A positive conclusion was made for this patient due to 2 out of the three trial tests conducted showing calculated concentrations closer to the calculated positive control. Despite a lack of strong florescence, in lab contamination is attributed to these anomalies.