BME100 s2016:Group13 W1030AM L5

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OUR TEAM

Name: Shantelle Woody
Name: Najla Alamro
Name: Bradley Taylor
Name: Andrej
Name: Slade
Name: Brian


LAB 5 WRITE-UP

PCR Reaction Report

The purpose of the PCR reaction was to test for the presence of dsDNA in the samples from two patients. The following materials from lab A were needed in order to perform the PCR reaction. First we cut the strip of eight PCR tubes into two strips of four test tubes, this was done in order to fit the test tubes into the PCR machine. After that the test tubes were labeled with a sharpie in order to indicate the identity of the tube. The identity of the tube was very important to keep track of because if the tube was mislabeled it would of caused inaccurate results. The test tubes were labeled as follows G13 +, G13 -, G13 1-1, G13 1-2, G13 1-3, G13 2-1, G13 2-2, and G13 2-3. The group number was labeled because two groups used one PCR machine and the patient and DNA sample number needed to be labeled in order to have accurate results. After that 50 micro-leters of PCR mix was pipetted into each test tube by going to the first stop outside of the test tube. Then placing it inside the test tube then releasing the stop removing the pipette and finally injecting to the second stop into the desired test tube. After each addition the tip was discarded into the waste cup and a new tip was placed on. After that 50 micro-liters of the positive control was pipetted into the positive control test tube creating a solution of 100 micro-liters. This was then done for the negative control, patient 1 replicates 1-3, and patient 2 replicates 1-3. The resulting solution for each test tube was then 100 micro-liters. The eight test tubes were then placed into the PCR machine, the OpenPCR program was then set, the lid was closed and the reaction began.

Fluorimeter Procedure

Smart phone set-up

For this experiment our group used a smart phone to take images of the PCR drop. First a group member with the best camera on their phone was chosen to be the photographer. The phone's settings were changed to:

  • flash was first inactivated
  • ISO to 800 or higher
  • White balance on auto
  • Exposure on highest setting
  • Saturation on the highest setting
  • Contrast to lowest setting

After the settings were changed the camera was placed facing the long side of the glass slide in order to get the image of the drop being illuminated. The stand that held it kept the phone at an angle that made the camera 4cm away from the glass slide. The distance of the camera to the drop was kept constant for the entire experiment.

Placing Samples onto the Fluorimeter

  1. Make sure all required materials are set and ready for use to complete the experiment. Set the mircopipette device to a volume of 80 micro-liters. Before beginning label all 8 empty tubes appropriately with one labeled for positive control and another for negative control. The remaining 6 split in half between two patients, patient 1 and patient 2.
  2. Using the 8 empty tubes
  3. Place a sterile tip onto the pipette
  4. Go to the first stop of the pipette and place it into the solution of 5 micrograms/mL of Calf thymus DNA solution and release.
  5. Place the pipette tip onto the surface of the plate in between the dots and go to the first stop in a controlled manner in order to eject the solution to form a drop.
  6. Discard the tip into the disposable waste container.
  7. Place a new tip onto the pipette.
  8. Go to first stop of the pipette and place it into the solution of SYBR Green I, and release.
  9. Take the pipette out and go to first stop in a controlled manner over the bubble in order to inject 80 micro-liters into the bubble forming a 160 micro-liter bubble. Then discard the tip.
  10. Place the box over the light box and phone and close the lid.
  11. Make sure the phone camera is 4cm away from the bubble. Then take three pictures of the bubble.
  12. Remove the bubble and move the glass slide over to the next gap.
  13. Repeat this procedure for the 2, 1, 0.5, 0.25, and 0 micrograms/mL of Calf Thymus DNA solution.
  14. The same procedure was adopted for the PCR which consisted of the eight test tubes containing the positive control, negative control, the three samples from patient one, and the three samples form patient two. 100 micro-liters of each marked test tube of PCR was placed into there respected test tubes contianing 500 micro-liters of buffer in order to insure that we would not run out of PCR. Hence, the SYBR Green I was injected to form a bubble and the PCR was injected into it creating a 160 micro-liter bubble. Also a new tip was used for every injection of PCR into their respected test tubes containing buffers. Also, a new tip was used for every injection in order to form a bubble.


Data Collection and Analysis

Images of High, Low, and Zero Calf Thymus DNA

High Calf Thymus

Highcal2.jpg

Low Calf Thymus

Lowcal2.jpg

Zero Calf Thymus

0cal2.jpg




Calibration Means

Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL) Final DNA concentration in SYBR Green I solution (µg/mL) Sample Number RAWINTDEN DROP - BACKGROUND MEAN Standard Deviation
Image 1 Image 2 Image 3
5 2.5 C-1 10058566 8150502 9973892 9394320 1078009.66
2 1 C-2 9104762 7915387 9075701 8698616.67 678452.407
1 0.5 C-3 6772632 4462128 6598888 5944549.33 1286750.36
0.5 0.25 C-4 4644319 3367472 4928435 4313408.67 831431.061
0.25 0.125 C-5 5802980 4118776 5800737 5240831 971728.782
0 0 C-6 3389555 2111190 3455391 2985378.67 757784.906

Calibration curves

Calibration Curve One

Group13 Lab5 CalibrationPlot1.PNG

Calibration Curve Two

Group13 Lab5 CalibrationPlot2.PNG



Images of Our PCR Negative and Positive Controls

Positive Control

PCRPOS.jpg

Negative Control

PCRNEG.jpg

PCR Results: PCR concentrations solved

PCR Concentrations Table

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)
G13 - 3847616.667 0.065704304 600 39.42258257
G13 + 9119084.333 1.091823638 600 655.0941828
G13 1-1 10822421.67 1.423387347 600 854.032408
G13 1-2 10176771.33 1.297708061 600 778.6248367
G13 1-3 8323650.333 0.936988162 600 562.1928971
G13 2-1 3954573.333 0.086523991 600 51.9143944
G13 2-2 3344661.333 -0.032198637 600 -19.31918244
G13 2-3 3231992 -0.054130325 600 -32.4781949




PCR Results: Summary

  • Our positive control PCR result was 655.09 μg/mL
  • Our negative control PCR result was 39.42 μg/mL


Observed results

Replicate 1 Replicate 2 Replicate 3 Qualitative
Patient 1 854.032408 778.6248367 562.1928971 The green spot was very noticable.
Patient 2 51.9143944 -19.31918244 -32.4781949 There was hardly any green spot.


Conclusions

  • Patient 1 : Positive because the average of all three replicates was above the 655.09μg/mL threshold established in the positive control.
  • Patient 2 : Negative because the average of all three replicates was below the 39.42μg/mL threshold established by the negative control.