BME100 s2017:Group3 W1030AM L5: Difference between revisions
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# Collect and label all necessary samples. | # Collect and label all necessary samples. | ||
# Add 80 microliters of SYBR Green to the rougher side of the glass slide. It will appear as a bubble. | # Add 80 microliters of SYBR Green to the rougher side of the glass slide between the first two rows. It will appear as a bubble. | ||
# Discard the pipette tip and replace it. | # Discard the pipette tip and replace it. | ||
# Add 80 microliters of one of the Calf Thyme samples to the bubble of SYBR Green. | # Add 80 microliters of one of the Calf Thyme samples to the bubble of SYBR Green. | ||
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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:5_Concentration_Photo.png|400px]]<br> | |||
5 μg/mL sample<br><br> | |||
[[Image:.5_Concentration_Photo.png|400px]]<br> | |||
.5 μg/mL sample<br><br> | |||
[[Image:0_Concentration_Photo.png|400px]]]<br> | |||
0 μg/mL sample | |||
'''Calibrator Mean Values''' | '''Calibrator Mean Values''' | ||
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[[Image:Table_2.png|800px]] | |||
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<!-- INSTRUCTIONS: Place images of your Excel plots (2 total) here. --> | <!-- INSTRUCTIONS: Place images of your Excel plots (2 total) here. --> | ||
[[Image:Excel_Plots.png|800x]] | |||
'''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. --> | ||
[[Image:Positive_photo.png|400px]]<br> | |||
positive control PCR sample<br><br> | |||
[[Image:Negative_photo.png|400px]]<br> | |||
negative control PCR sample | |||
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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. --> | ||
[[Image:Table_5.png|800x]] | |||
'''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 -1.827429919 μg/mL | ||
* Our negative control PCR result was | * Our negative control PCR result was -47.11443396 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 14149 : | ||
* Patient | Qualitative - The image of patient 14149's sample appeared clear and showed no green color. | ||
Quantitative - The initial PCR product concentrations of patient 14149's sample was shown to be -47.09143846μg/mL, 5.820222019μg/mL, 5.810535581μg/mL. | |||
* Patient 42390 : | |||
Qualitative - The image of patient 42390's sample appeared clear and showed no green color. | |||
Quantitative - The initial PCR product concentrations of patient 42390's sample was shown to be 2.500624361μg/mL, -17.13046712μg/mL, -1.465672039μg/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 | * Patient 14149 negative : Both the qualitative description and quantitative data showed that the sample was negative. The negative control appeared clear and had no green color which was similar to patient 14149's sample. Also the initial PCR product concentration of patient 14149's 1st sample (-47.09143846μg/mL) was similar to the negative control's concentration (-47.11443396 μg/mL). The other two samples were inconclusive. | ||
* Patient | |||
* Patient 42390 negative :Both the qualitative description and quantitative data showed that the sample was negative. The negative control appeared clear and had no green color which was similar to patient 42390's sample. Also the concentration of patient 42390's 2nd sample (-17.13046712μg/mL) was closest to the initial PCR product concentration of the negative control's concentration (-47.11443396 μg/mL). While the third sample's initial PCR product concentration (-1.465672039μg/mL) was similar to the positive's (-1.827429919 μg/mL) the visual aspect pushes more for a nagative sample. | |||
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'''Gel Electrophoresis''' | '''Gel Electrophoresis''' | ||
[[Image:Gel electrophoresis.PNG|600px]] | |||
From left to right: | |||
# Ladder | |||
# Positive Control | |||
# Negative Control | |||
# Patient 1-1 | |||
# Patient 1-2 | |||
# Patient 1-3 | |||
# Patient 2-1 | |||
# Patient 2-2 | |||
# Patient 2-3 | |||
<!-- Do not edit below this line --> | <!-- Do not edit below this line --> | ||
|} | |} |
Latest revision as of 20:48, 4 April 2017
BME 100 Spring 2017 | 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 ReportUsing new equipment for the first time was difficult but the pre-lab gave easy to understand instructions. It took some time for us to understand the difference between the first and second stop but by looking at the directions we were able to figure it out. The first stop takes in the liquid and the second stop releases it. Between the two stops we could feel a click within the pipette. Once we were done mixing the controls and patients tests there was a trace amount of DNA samples and PCR reaction mix left within their tubes. Because there was small varying amounts of liquid left of the DNA samples and PCR reaction mix the final reactions did not have exactly the same amount of liquid. With more practice using the pipette it would be easier to leave less amount of DNA samples and PCR reaction mix and have the same amount of liquid in the final reactions. During the experiment we did not have to change our labeling scheme, we used the same scheme that we planned to use. Fluorimeter ProcedureImaging set-up
Data Collection and AnalysisImages of High, Low, and Zero Calf Thymus DNA Calibrator Mean Values
Images of Our PCR Negative and Positive Controls
Qualitative - The image of patient 14149's sample appeared clear and showed no green color. Quantitative - The initial PCR product concentrations of patient 14149's sample was shown to be -47.09143846μg/mL, 5.820222019μg/mL, 5.810535581μg/mL.
Qualitative - The image of patient 42390's sample appeared clear and showed no green color. Quantitative - The initial PCR product concentrations of patient 42390's sample was shown to be 2.500624361μg/mL, -17.13046712μg/mL, -1.465672039μg/mL. Conclusions
Gel Electrophoresis From left to right:
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