BME100 f2016:Group10 W1030AM L5: Difference between revisions
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'''Placing Samples onto the Fluorimeter''' | '''Placing Samples onto the Fluorimeter''' | ||
<!-- 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 --> | ||
Procedure | |||
Calibration procedure | |||
==Calibration Procedure== | |||
<br>1. A fluorimeter was placed on a flat hard surface adjusting the height with stacked objects to accommodate the height of the smartphone camera | |||
<br>2. Using gloves, one slide was placed into the fluorimeter with the smooth side down. | |||
<br>3. The smart phone camera app was initiated, set to an 8 sec shutter release and placed into the cradle. | |||
<br>4. The camera and fluorimeter were adjusted to get a horizontal edge-on view of the slide. | |||
<br>5. The micro-pipette was set to 85 µL to ensure the full 80 µL of solutions were added. | |||
<br>6. A clean pipette tip was pressed onto the micro-pipette. | |||
<br>7. The micro-pipette was pushed to the first position dispensing 80 µL of SYBR Green I solution onto the slide to form a beach ball shaped droplet on the first 2 circles on the slide. | |||
<br>8. The micro-pipette tip was discarded into the disposal cup. | |||
<br>9. A new tip was placed onto the micro-pipette and 80 µL of DNA thymus solution was added to the center of the SYBR Green droplet. | |||
<br>10. The blue light was turned on, the slide was adjusted to illuminate the center of the drop and through the other side. | |||
<br>11. The distance from the smartphone camera lens and the fluorimeter were adjusted to an average distance of 8cm. | |||
<br>12. The fluorimeter and the smartphone were covered with a light box, one side of the light box was left open the flash was turned off and the focus adjusted. | |||
<br>13. The camera timer was initiated and the light box flap was lowered to ensure darkness within the light box and the picture was taken. | |||
<br>14. After the photo was checked to ensure clarity, the 160 µL drop was removed from the slide. | |||
<br>15. The slide was then advanced to the next position ensuring no solution residue was present at that slide location. | |||
<br>16. These subsequent steps were repeated for all 5 Calf Thymus DNA solutions, taking three pictures of each for a total of 15 photos for the calibration section. | |||
==PCR Reaction Procedure== | |||
<br>1. Obtain PCR reaction samples from instructors. | |||
<br>2. Each RED DOT (buffer) tube was labeled to match the 8 PCR reaction samples created in part C. | |||
<br>3. The micro pipette was then set tp0 100 micro liters to ensure a full draw of 100 micro liters o the PCR sample. | |||
<br>4. Using a new tip, the 100 micro liters of PCR sample were placed into the buffer solution tube with the duplicate label. | |||
<br>5. This was done for all 8 samples refreshing the tip between each transfer. | |||
<br>6. The caps of each tube were secured and the tube was inverted to ensure the buffer DNA mix. | |||
<br>7. Steps 1-16 used for the calibration samples were repeated with the SYBR GREEN I solution and the PCR reaction/Buffer solution. | |||
<br> | <br> | ||
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==Data Collection and Analysis== | ==Data Collection and Analysis== | ||
'''Images of High, Low, and Zero Calf Thymus DNA''' | '''Images of High, Low, and Zero Calf Thymus DNA'''<br> | ||
<!-- 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. --> | ||
[[Image:HighConcentration.PNG|200px|High Concentration]] 5 μg/mL DNA sample <br> | |||
[[Image:MiddleConcentration.PNG|200px|Middle Concentration]] 0.5 μg/mL DNA sample <br> | |||
[[Image:LowConcentration.PNG|200px|Low Concentration]] 0 μg/mL DNA sample<br> | |||
'''Calibrator Mean Values''' | '''Calibrator Mean Values''' | ||
<!-- INSTRUCTIONS: Show all values from Excel Table 2 from Section 3. '''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 2 from Section 3. '''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. --> | ||
{| {{table}} | |||
|- | |||
| || || || '''RAWINTDEN DROP-BACKGROUND''' ||'''RAWINTDEN DROP-BACKGROUND''' ||'''RAWINTDEN DROP-BACKGROUND'''|| || | |||
|- | |||
| '''Initial Concentration of 2X Calf Thymus DNA solution (micrograms/mL)''' || '''Final DNA concentration in SYBR Green I solution (µg/mL)''' || '''Sample Number''' || '''Image 1''' || '''Image 2''' || '''Image 3''' || '''Mean''' || '''Standard Deviation''' | |||
|- | |||
| 5 || 2.5 || C-1 || 62340563 || 56779681 || 28365187 || 49161810.33 || 18223762.2 | |||
|- | |||
| 2 || 1 || C-2 || 31023532 || 31570307 || 32750900 || 31781579.67 || 882851.72 | |||
|- | |||
| 1 || 0.5 || C-3 || 29342990 || 27835695 || 19230059 || 25469581.33 || 5455887.99 | |||
|- | |||
| 0.5 || 0.25 || C-4 || 42475213 || 2863945 || 66503660 || 37280939.33 || 32136251.97 | |||
|- | |||
| 0.25 || 0.125 || C-5 || 11652572 || 15057462 || 19458988 || 15389674 || 3913796.92 | |||
|- | |||
| 0 || 0 || C-6 || 11600278 || 12714394 || 13923961 || 12746211 || 1162168.19 | |||
|} | |||
'''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:Lab_5_pic_1.PNG|400px|Plot 1]] | |||
[[Image:Lab_5_pic_2.PNG|400px|Plot 2]] | |||
'''Images of Our PCR Negative and Positive Controls''' <br> | |||
'''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. --> | ||
[[Image:PositivePic.PNG|200px|Low Concentration]] Positive Control PCR Sample <br> | |||
[[Image:NegativePic.PNG|200px|Low Concentration]] Negative Control PCR Sample <br> | |||
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<!-- 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. --> | ||
{| {{table}} | |||
|- | |||
| '''PCR Product TUBE LABEL''' || '''MEAN (of RAWINTDEN DROP - BACKGROUND)''' || '''PCR Product Concentration (µg /mL)''' || '''Total Dilution (µL)''' || '''Initial PCR Product Concentration''' | |||
|- | |||
| Positive|| 9431671.667 || -1.056832833 || 0.08|| -0.088069403 | |||
|- | |||
| Negative||4796648.667 ||-1.520335133 || 0.08|| -0.126694594 | |||
|- | |||
| Patient 1-1|| 6975292 || -1.3024708 ||0.08|| -0.108539233 | |||
|- | |||
| Patient 1-2||5522674.667 ||-1.447732533 || 0.08|| -0.120644378 | |||
|- | |||
| Patient 1-3|| 12002167.33 ||-0.799783267 || 0.08|| -0.066648606 | |||
|- | |||
| Patient 2-1||7843444.667 ||-1.215655533 || 0.08|| -0.101304628 | |||
|- | |||
| Patient 2-2|| 41200677.33 ||2.120067733 || 0.08|| 0.176672311 | |||
|- | |||
| Patient 2-3|| 10835099 ||-0.9164901 || 0.08|| -0.076374175 | |||
|} | |||
'''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 -0.088069403 μg/mL | ||
* Our negative control PCR result was | * Our negative control PCR result was -0.126694594 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 77434 : | * Patient 77434 : All three images looked blurry and bright, similar to our negative control PCR sample. The initial PCR product concentration for Patient 77434 were measured as -0.108539233 μg/mL, -0.120644378 μg/mL, and -0.066648606 μg/mL. | ||
* Patient 77218 : | * Patient 77218 : Two of the three images looked blurry and bright, similar to our negative control PCR sample. However, the third one looked more clear like the postive control PCR sample image. The initial PCR product concentration for Patient 77218 were measured as -0.101304628 μg/mL, 0.176672311 μg/mL, and -0.076374175 μg/mL. | ||
<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 77434 : | * Patient 77434 : The patient's values were close to the negative PCR results, therefore, we concluded that patient 77434 was negative. | ||
* Patient 77218 : | * Patient 77218 : The patient's values were sporadic but closer to positive PCR results, therefore, we concluded that patient 77218 was positive. ('''Note''': Although we said Patient 77218 was positive, The Gold Standard labeled the patient negative.) | ||
<!-- Do not edit below this line --> | <!-- Do not edit below this line --> | ||
|} | |} |
Latest revision as of 12:28, 2 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 pre-lab reading was very interactive and informative. For those who have never used a micropipette, the pre-lab information was beneficial. The first stop on the pipette is used to collect the sample that is being transferred and the second stop is used to release the sample liquid. The final reaction will have the same amount of liquid. There was no liquids left in the tubes of the DNA samples and PCR reaction mix. Fluorimeter ProcedureImaging set-up A member in our group provided their iPhone 6s camera in order to take images while the fluorimeter measures the fluorescence of the DNA. This is the transmittance of the wavelength given off by the DNA that is excited. The wavelength of light is exciting the DNA and if it fluoresces then there is double-stranded DNA present. Taking the iPhone 6s, the camera's flash was inactived in order to get a reasonable image of the calibration with the DNA concentration. The iPhone 6s was placed on to a metal card holder and the metal card holder was put on top of a plastic stand at a distance of 4 centimeters. The iPhone 6s was leaning on an angle due to the metal card holder and we wanted to have the camera perpendicular to the fluorimeter. In order to do this, we placed a folded piece of paper behind the iPhone 6s to prop it up. Placing Samples onto the Fluorimeter Procedure Calibration procedure
Calibration Procedure
PCR Reaction Procedure
Data Collection and AnalysisImages of High, Low, and Zero Calf Thymus DNA Calibrator Mean Values
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