BME100 f2016:Group15 W8AM L5: Difference between revisions
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{| style="wikitable" width="700px" | {| style="wikitable" width="700px" | ||
|- valign="top" | |- valign="top" | ||
| [[Image: | | [[Image:20160907 184548.jpg|100px|thumb|Name: Adam Akkad]] | ||
| [[Image: | | [[Image:IMG_0461.JPG|100px|thumb|Name: Shad Boswell]] | ||
| [[ | | [[image:IMG_0854-1.jpg|100px|thumb|Name: Nathan Hui]] | ||
| [[ | | [[image:IMG_0856.jpg|100px|thumb|Name: Carli Winchester]] | ||
|} | |} | ||
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==PCR Reaction Report== | ==PCR Reaction Report== | ||
<!-- Write a summary of your team's experience with pipetting the samples to set up the reaction. Did the pre-lab reading help you? Did you understand the difference between the first and second stop on the pipettor? Did the final reactions have exactly the same amount of liquid? Was there any liquid left in the tubes that the DNA samples and PCR reaction mix? Did you have to change your labeling scheme? --> | <!-- Write a summary of your team's experience with pipetting the samples to set up the reaction. Did the pre-lab reading help you? Did you understand the difference between the first and second stop on the pipettor? Did the final reactions have exactly the same amount of liquid? Was there any liquid left in the tubes that the DNA samples and PCR reaction mix? Did you have to change your labeling scheme? --> | ||
The pipetting and PCR process was relatively smooth. We understood the that the difference between the first and second stop on the pipettor was that the first stop is used to absorb the liquid into the pipettor and the second stop is pressed to eject the liquid out. Our final reactions had the same amount of liquid as before the reaction. There was no liquid left in the DNA samples or PCR reaction mix. Our labeling scheme was consistent--it did not require any changes. | |||
==Fluorimeter Procedure== | ==Fluorimeter Procedure== | ||
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<!-- INSTRUCTIONS: In the space below, describe in detail how your team set up your device to capture images from the fluorimeter. --> | <!-- INSTRUCTIONS: In the space below, describe in detail how your team set up your device to capture images from the fluorimeter. --> | ||
First, we set up our smartphone camera vertically and placed it in the camera holder provided for us. We placed the slide onto the fluorimeter with the smooth side down. We then elevated the fluorimeter using several trays so that the camera was level with the slide on the fluorimeter. The camera was set to a timer of 5 seconds. After pipetting the fluorescent liquid and the solution we were measuring onto the slide, we took the picture and covered the phone and fluorimeter with a box so the picture could see the fluorescence more clearly. | |||
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<!-- INSTRUCTIONS: In the space below, in your own words write the steps you performed to place samples onto the fluorimeter --> | <!-- INSTRUCTIONS: In the space below, in your own words write the steps you performed to place samples onto the fluorimeter --> | ||
It should first be noted that all of the drops must be placed on a fluorimeter slide. In order to ensure that proper bubbles are formed, the rough side of the slide should be the side facing upwards. Beginning with the SYBR GREEN 1 solution, we added 80 uL to the rough side of the slide. Following this, 80 uL of H2O were added right on top of the SYBR GREEN 1 solution. This gave us a clear bubble which we could analyze. The blue LED light was then turned on. Adjustments were made to make sure that the light went directly through the middle of the solution bubble. The box was lowered to cover the fluorimeter to allow for proper lighting, after which 3 separate images were taken of the solution bubble. This process was repeated for all of the Calf Thymus DNA concentrations, as well as the PCR DNA mix from the previous lab. | |||
<br> | <br> | ||
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'''Images of High, Low, and Zero Calf Thymus DNA''' | '''Images of High, Low, and Zero Calf Thymus DNA''' | ||
<!-- INSTRUCTIONS: Show ONE image where you drew a circle around the droplet in ImageJ for any image for the (1) 5 μg/mL sample (2) 0.5 μg/mL sample and (3) zero DNA. Please crop your images so that only the drop and a small empty rectangular region around the drop are included. Lots of empty space is a waste of space. --> | <!-- INSTRUCTIONS: Show ONE image where you drew a circle around the droplet in ImageJ for any image for the (1) 5 μg/mL sample (2) 0.5 μg/mL sample and (3) zero DNA. Please crop your images so that only the drop and a small empty rectangular region around the drop are included. Lots of empty space is a waste of space. --> | ||
1. 5 μg/mL sample [[Image:Screen Shot 2016-11-01 at 6.32.18 PM]] | |||
2. 0.5 μg/mL sample [[Image:Screen Shot 2016-11-01 at 6.31.28 PM]] | |||
3. zero DNA [[Image:Screen Shot 2016-11-01 at 6.33.18 PM]] | |||
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{| class="wikitable" | |||
! 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 | |||
| 66323 | |||
| 66323 | |||
| 66323 | |||
| 66323 | |||
| 0 | |||
|- | |||
| 2 | |||
| 1 | |||
| C-2 | |||
| 286393 | |||
| 286393 | |||
| 286393 | |||
| 286393 | |||
| 0 | |||
|- | |||
| 1 | |||
| 0.5 | |||
| C-3 | |||
| 327772 | |||
| 327772 | |||
| 327772 | |||
| 327772 | |||
| 0 | |||
|- | |||
| 0.5 | |||
| 0.25 | |||
| C-4 | |||
| 341592 | |||
| 341592 | |||
| 341592 | |||
| 341592 | |||
| 0 | |||
|- | |||
| 0.25 | |||
| 0.125 | |||
| C-5 | |||
| 280596 | |||
| 280596 | |||
| 280596 | |||
| 280596 | |||
| 0 | |||
|- | |||
| 0 | |||
| 0 | |||
| C-6 | |||
| 302079 | |||
| 302079 | |||
| 302079 | |||
| 302079 | |||
| 0 | |||
|} | |||
'''Calibration curves'''<br> | '''Calibration curves'''<br> | ||
<!-- INSTRUCTIONS: Place images of your Excel plots (2 total) here. --> | <!-- INSTRUCTIONS: Place images of your Excel plots (2 total) here. --> | ||
[[Image:Screenshot 2016-11-01 at 9.11.56 PM.png]] | |||
'''Images of Our PCR Negative and Positive Controls''' | '''Images of Our PCR Negative and Positive Controls''' | ||
<!-- INSTRUCTIONS: Show ONE image where you drew a circle around the droplet in ImageJ for any image for the (1) Negative control PCR sample AND (2) the Positive control PCR sample. --> | <!-- INSTRUCTIONS: Show ONE image where you drew a circle around the droplet in ImageJ for any image for the (1) Negative control PCR sample AND (2) the Positive control PCR sample. --> | ||
*Positive | |||
[[Image:Screenshot 2016-11-01 at 10.38.21 PM.png]] | |||
*Negative | |||
[[Image:Screenshot 2016-11-01 at 10.40.35 PM.png]] | |||
'''PCR Results: PCR concentrations solved''' | '''PCR Results: PCR concentrations solved''' | ||
<!-- INSTRUCTIONS: Show all values from Excel Table 5 from Section 5. '''To save time on typing a new Wiki table from scratch''', use THIS TOOL to auto-generate a Wiki table: http://excel2wiki.net/wikipedia.php. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code. --> | <!-- INSTRUCTIONS: Show all values from Excel Table 5 from Section 5. '''To save time on typing a new Wiki table from scratch''', use THIS TOOL to auto-generate a Wiki table: http://excel2wiki.net/wikipedia.php. Copy the headers and values from the Excel spreadsheet you made, paste them into the form field, click submit, copy the Wiki code that the tool generated, and replace TABLE GOES HERE (below) with your auto-generated code. --> | ||
{| class="wikitable" | |||
! 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) | |||
|- | |||
| Patient 1.1 | |||
| 435207 | |||
| 1.937217099 | |||
| 12 | |||
| 23.24660518 | |||
|- | |||
| Patient 1.2 | |||
| 558815 | |||
| 4.439181753 | |||
| 12 | |||
| 53.27018103 | |||
|- | |||
| Patient 1.3 | |||
| 588523 | |||
| 5.040505016 | |||
| 12 | |||
| 60.48606019 | |||
|- | |||
| Negative | |||
| 473517 | |||
| 2.712654497 | |||
| 12 | |||
| 32.55185396 | |||
|- | |||
| Patient 2.1 | |||
| 1399455 | |||
| 21.45467886 | |||
| 12 | |||
| 257.4561463 | |||
|- | |||
| Patient 2.2 | |||
| 1183939 | |||
| 17.09239313 | |||
| 12 | |||
| 205.1087176 | |||
|- | |||
| Patient 2.3 | |||
| 1264326 | |||
| 18.71951624 | |||
| 12 | |||
| 224.6341948 | |||
|- | |||
| Positive | |||
| 1257350 | |||
| 18.57831417 | |||
| 12 | |||
| 222.93977 | |||
|} | |||
'''PCR Results: Summary''' | '''PCR Results: Summary''' | ||
<!-- INSTRUCTIONS: You completed 8 PCR reactions and used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as '''threshold''' values for determining whether an unknown (patient) sample is truly positive or negative. Replace the underscore with your calculated initial concentration values.--> | <!-- INSTRUCTIONS: You completed 8 PCR reactions and used the SYBR Green I staining and imaging technique to measure the amount of amplified DNA in each PCR reaction. You used a standard curve (based on known concentrations of calf thymus DNA) to convert INTDEN values into DNA concentration. Your positive control and negative control samples should be used as '''threshold''' values for determining whether an unknown (patient) sample is truly positive or negative. Replace the underscore with your calculated initial concentration values.--> | ||
* Our positive control PCR result was | * Our positive control PCR result was 222.93977 μg/mL | ||
* Our negative control PCR result was | * Our negative control PCR result was 32.55185396 μg/mL | ||
<u>Observed results</u> | <u>Observed results</u> | ||
<!-- INSTRUCTIONS: Replace the underscore with each patient ID. After the colon, write both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed --> | <!-- INSTRUCTIONS: Replace the underscore with each patient ID. After the colon, write both a qualitative (what the images looked like) and a quantitative description (μg/mL) of what you observed --> | ||
* Patient | * Patient 1 : The images did not fluoresce very much. Not very much green color. The initial PCR Product Concentration was 23.24660518,53.27018103, 60.48606019 respectively for each of the three trials of patient 1. | ||
* Patient | * Patient 2 : The images all fluoresced bright green. The initial PCR Product Concentration was 257.4561463,205.1087176, 224.6341948 respectively for each of the three trials of patient 2. | ||
<u>Conclusions</u> | <u>Conclusions</u> | ||
<!-- INSTRUCTIONS: Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion. --> | <!-- INSTRUCTIONS: Compare each patient's results to the positive control value and the negative control value. Draw a final conclusion for each patient (positive or negative) and explain why you made that conclusion. --> | ||
* Patient | * Patient 1 : Values for the initial concentration were closest to the negative control.Therefore, this patient tests negative for the diseased DNA. | ||
* Patient | * Patient 2 : Values for the initial concentration were closest to the positive control.Therefore, this patient tests positive for the diseased DNA. | ||
Latest revision as of 23:51, 1 November 2016
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 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAM
LAB 5 WRITE-UPPCR Reaction ReportThe pipetting and PCR process was relatively smooth. We understood the that the difference between the first and second stop on the pipettor was that the first stop is used to absorb the liquid into the pipettor and the second stop is pressed to eject the liquid out. Our final reactions had the same amount of liquid as before the reaction. There was no liquid left in the DNA samples or PCR reaction mix. Our labeling scheme was consistent--it did not require any changes. Fluorimeter ProcedureImaging set-up First, we set up our smartphone camera vertically and placed it in the camera holder provided for us. We placed the slide onto the fluorimeter with the smooth side down. We then elevated the fluorimeter using several trays so that the camera was level with the slide on the fluorimeter. The camera was set to a timer of 5 seconds. After pipetting the fluorescent liquid and the solution we were measuring onto the slide, we took the picture and covered the phone and fluorimeter with a box so the picture could see the fluorescence more clearly.
Placing Samples onto the Fluorimeter It should first be noted that all of the drops must be placed on a fluorimeter slide. In order to ensure that proper bubbles are formed, the rough side of the slide should be the side facing upwards. Beginning with the SYBR GREEN 1 solution, we added 80 uL to the rough side of the slide. Following this, 80 uL of H2O were added right on top of the SYBR GREEN 1 solution. This gave us a clear bubble which we could analyze. The blue LED light was then turned on. Adjustments were made to make sure that the light went directly through the middle of the solution bubble. The box was lowered to cover the fluorimeter to allow for proper lighting, after which 3 separate images were taken of the solution bubble. This process was repeated for all of the Calf Thymus DNA concentrations, as well as the PCR DNA mix from the previous lab.
Data Collection and AnalysisImages of High, Low, and Zero Calf Thymus DNA
Calibration curves Images of Our PCR Negative and Positive Controls
PCR Results: PCR concentrations solved
PCR Results: Summary
|