BME100 f2014:Group4 L5: Difference between revisions
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|- valign="top" | |- valign="top" | ||
| [[Image:Twinsie.jpg|200px|thumb|Name: Anyssa Iwamoto]] | | [[Image:Twinsie.jpg|200px|thumb|Name: Anyssa Iwamoto]] | ||
| [[Image: | | [[Image:Imagge.jpeg|150px|thumb|Name: Shreya Ramkumar]] | ||
| [[Image: | | [[Image:DSC02469.jpg|150px|thumb|Name: Izzy Ortiz]] | ||
| [[Image: | | [[Image:Image_(1).jpeg|200px|thumb|Name: Larrison Black]] | ||
| [[Image: | | [[Image:10256865_472407076226016_945427824445492848_o_(1).jpg|150px|thumb|Name: Zach Sledge]] | ||
| [[Image: | | [[Image:1424344_653432334718295_1976595533_n_(1).jpg|150px|thumb|Name: Dominique Stewart]] | ||
|} | |} | ||
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'''Solutions Used for Calibration''' | '''Solutions Used for Calibration''' | ||
{| {{table}} width=700 | {| {{table}} width=700 | ||
|- | |- align="center" | ||
| | | Initial Concentration of 2X Calf Thymus DNA Solution (micrograms/mL) || Volume of the 2X DNA Solution (uL) || Volume of the SYBR Green I Solution (uL) || Final DNA Concentration in SYBR Green I Solution (ug/mL) | ||
|- | |- align="center" | ||
| | | 5 || 80 || 80 || 2.5 | ||
|- | |- align="center" | ||
| | | 2 || 80 || 80 || 1 | ||
|- align="center" | |||
| 1 || 80 || 80 || 0.5 | |||
|- align="center" | |||
| 0.5 || 80 || 80 || 0.25 | |||
|- align="center" | |||
| 0.25 || 80 || 80 || 0.125 | |||
|- align="center" | |||
| 0 || 80 || 80 || 0 | |||
|} | |} | ||
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'''Placing Samples onto the Fluorimeter''' | '''Placing Samples onto the Fluorimeter''' | ||
# | # Slide the glass slide onto the fluorimeter with the smooth side down and adjust the glass so that the blue light is shining in between two white circles. | ||
# | # Set up the phone camera to the three second timer and turn the flash off. (If the flash is not able to be disabled, skip this step) | ||
# Place the phone in the holder and make sure the glass slide is level with the camera and in focus. | |||
# Measure the distance between the phone and the fluorimeter (Make sure the camera is more than 4 cm away, in our experiment we used 7.8cm) | |||
# Place 80 uL drops of SYBR Green I solution in the between the two circles that the blue light is illuminating. (Remember to use a new micropipette tip for each different solution and discarding the tips in the red disposable cup.) | |||
# Place 80 uL drops of the sample solution onto the SYBR Green I drop that is already on the glass slide. | |||
# Place the box over the fluorimeter with the opening on the side of the camera. | |||
# Focus the image and click on the timer before covering the box with the lid OR cover the box as much as possible while clicking the shutter manually. | |||
# Using the micropipette, remove all 160 uL solution from the slide and discard the solution into the red disposable cup. | |||
# Move the slide so the blue light shines between two new white circles. | |||
# Repeat this procedure for each sample three times. | |||
Put the glass slide in the sharps container once all the possible white circle have been used. | |||
'''Bonus: Images of Setup''' | |||
[[Image:Image(2).jpeg|500px|Desc]] | |||
Above is an image of the basic setup without the black box over it. Below is an image of the setup with the black box over it but without closing the final side so that we could take the picture. | |||
[[Image:Image_(3).jpeg|500px|Desc]] | |||
<br> | <br> | ||
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'''Representative Images of Negative and Positive Samples''' | '''Representative Images of Negative and Positive Samples''' | ||
<!-- INSTRUCTIONS: (1) Show ONE image where you drew a circle around the droplet with the freehand tool for any sample with *no* DNA. (2) Show ONE image where you drew a circle around the droplet with the freehand tool for a sample *with* DNA (positive signal). -If you include more than two images, you will not receive any additional credit. --> | <!-- INSTRUCTIONS: (1) Show ONE image where you drew a circle around the droplet with the freehand tool for any sample with *no* DNA. (2) Show ONE image where you drew a circle around the droplet with the freehand tool for a sample *with* DNA (positive signal). -If you include more than two images, you will not receive any additional credit. --> | ||
'''Negative Sample''' [[Image:Negative_G4.JPG|400px|Desc]] '''Positive Sample''' [[Image:Positive_G4.JPG|400px|Desc]] | |||
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<!-- INSTRUCTIONS: Show a table for the ImageJ calf thymus DNA data. '''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 a table for the ImageJ calf thymus DNA data. '''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}} width=700 | |||
| align="center" style="background:#f0f0f0;"|'''PCR Product Label Tube''' | |||
| align="center" style="background:#f0f0f0;"|'''Area''' | |||
| align="center" style="background:#f0f0f0;"|'''Mean Pixel Value''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN of Drop''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN of Background''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTEDEN (Drop-Background)''' | |||
|- align="center" | |||
| 5||498||238.514||118780||755||118025 | |||
|- align="center" | |||
| 5||498||237.96||118504||1135||117369 | |||
|- align="center" | |||
| 5||498||253.303||126145||1918||124227 | |||
|- align="center" | |||
| 2||498||242.853||120941||750||120191 | |||
|- align="center" | |||
| 2||498||201.57||100382||659||99723 | |||
|- align="center" | |||
| 1||498||170.665||84991||687||84304 | |||
|- align="center" | |||
| 1||498||163.55||81448||1206||80242 | |||
|- align="center" | |||
| 1||498||171.643||85478||786||84692 | |||
|- align="center" | |||
| 0.5||498||115.305||57422||740||56682 | |||
|- align="center" | |||
| 0.5||500||113.478||56739||840||55899 | |||
|- align="center" | |||
| 0.5||484||113.291||54833||799||54034 | |||
|- align="center" | |||
| 0.25||484||68.285||33050||632||32418 | |||
|- align="center" | |||
| 0.25||484||76.911||34321||722||33599 | |||
|- align="center" | |||
| 0.25||484||70.155||33955||1465||32490 | |||
|- align="center" | |||
| 0||484||7.395||3579||982||2597 | |||
|- align="center" | |||
| 0||484||7.795||3773||693||3080 | |||
|- align="center" | |||
| 0||484||7.905||3826||565||3261 | |||
|- | |||
|} | |||
'''Image J Data''' | |||
{| {{table}} width=700 | {|{{table}} width=700 | ||
| | | align="center" style="background:#f0f0f0;"|'''PCR Product Label Tube''' | ||
| align="center" style="background:#f0f0f0;"|'''RAWINTEDEN (Drop-Background) AVERAGE''' | |||
| align="center" style="background:#f0f0f0;"|'''Standard Deviation''' | |||
|- align="center" | |||
|- | | 2.5||119873.67||3784.34 | ||
| | |- align="center" | ||
| 1||109957.00||14473.06 | |||
|- align="center" | |||
| 0.5||83079.33||2464.85 | |||
|- align="center" | |||
|- | | 0.25||55538.33||1360.34 | ||
| | |- align="center" | ||
| 0.125||32835.67||662.05 | |||
|- align="center" | |||
|- | | 0||2979.33||343.26 | ||
| | |||
|- | |||
| 0. | |||
|- | |||
| 0. | |||
|- | |||
| 0 | |||
|- | |- | ||
|} | |} | ||
'''Calibration curve'''<br> | '''Calibration curve'''<br> | ||
<!-- INSTRUCTIONS: Place an image of your Excel plot with a line of best fit here. --> | <!-- INSTRUCTIONS: Place an image of your Excel plot with a line of best fit here. --> | ||
[[Image:Curve1.JPG|600px|Desc]] | |||
<br>The last two values plateau in a non-linear way with the graph above throwing off the line of best fit which is why we excluded them on the graph below. | |||
<br> | |||
[[Image:Curve2.JPG|600px|Desc]] | |||
'''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 claculated 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 claculated initial concentration values.--> | ||
* Our positive control PCR result was | |||
* Our negative control PCR result was | {|{{table}} width=700 | ||
| align="center" style="background:#f0f0f0;"|'''PCR Product Label Tube''' | |||
| align="center" style="background:#f0f0f0;"|'''Average''' | |||
| align="center" style="background:#f0f0f0;"|'''PCR Production Concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''Initial PCR Production Concentration''' | |||
|- align="center" | |||
| G1-1||3964||-0.0349||-0.0029 | |||
|- align="center" | |||
| G1-2||2602.5||-0.0436||-0.0036 | |||
|- align="center" | |||
| G1-3||1945.5||-0.0478||-0.0040 | |||
|- align="center" | |||
| G2-3||5176||-0.0271||-0.0023 | |||
|- align="center" | |||
| G2-2||4825||-0.0294||-0.0024 | |||
|- align="center" | |||
| G2-3||3850||-0.0356||-0.0030 | |||
|- align="center" | |||
| Negative||82357.5||0.4666||'''0.0389''' | |||
|- align="center" | |||
| Positive||122055||0.7206||'''0.0600''' | |||
|- | |||
|} | |||
* Our positive control PCR result was 0.0600 μg/mL | |||
* Our negative control PCR result was 0.0389 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 85470 : The values on this patient were -.0029, -.0036, and .0040 μg/mL. The drops were not green at all so it was negative. | ||
* Patient | * Patient 90113 : The values on this patient were -.0023, -.0024, and .0030 μg/mL. The drops were not green at all so it was also negative. | ||
<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 85470 : The results suggest that Patient 85470 is closer to the negative value. | ||
* Patient | * Patient 90113 : The results suggest that Patient 90113 is closer to the negative value. | ||
<br> | |||
==SNP Information & Primer Design== | ==SNP Information & Primer Design== | ||
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'''Background: About the Disease SNP''' | '''Background: About the Disease SNP''' | ||
<!-- INSTRUCTIONS: This content is from PCR Lab D. Write a summary, at least five sentences long, about the disease SNP in your own words. --> | <!-- INSTRUCTIONS: This content is from PCR Lab D. Write a summary, at least five sentences long, about the disease SNP in your own words. --> | ||
<br> | |||
SNP is the abbreviation meaning Single Nucleotide Polymorphism and affect appearance, respond to drugs, and pre-disposition to diseases. In this lab, the particular SNP that is being analyzed is rs16991654, which is called Homo Sapiens (humans) in latin. On the National Center for Biotechnology (NCBI) page, numerous information on this SNP's can be found including the clinical significance, which is pathogenic or the disease linked to this SNP, which is Long QT syndrome (LQTS). The area of information that we focused on for the majority of the time is the gene sequence, which is KCNE2 where we analyzed the numerical position of the SNP (34370656) and had to not the non-disease forward primer, non-disease reverse primer, the disease forward primer, and the disease reverse primer. KCNE2 is gene coding which regulates numerous functions such as the neurotransmitter release, the heart rate, insulin secretion, neuronal excitability, and several others. | |||
{|{{table}} width=700 | |||
|- align="center" | |||
| Latin Name||Homo Sapien (Humans) | |||
|- align="center" | |||
| Chromosome||21:34370656 | |||
|- align="center" | |||
| Clinical Significance||Pathognic | |||
|- align="center" | |||
| Associated Gene||KCNE2 (Potassium Voltage-Gated Chanel, Isk-related family member) | |||
|- align="center" | |||
| Associated Disease||Long QT Syndrome (LQTS) | |||
|- align="center" | |||
| Numerical Postion||34370656 | |||
|- align="center" | |||
| Numerical Position (200 bases to the right)||34370856 | |||
|- align="center" | |||
| Non-Disease Forward Primer||CAT-GGT-GAT-GAT-TGG-AAT-GT | |||
|- align="center" | |||
| Non-Disease Reverse Primer||CCC-TTA-TCA-GGG-GGA-CAT-TT | |||
|- align="center" | |||
| Disease Forward Primer||CAT-GGT-GAT-GAT-TGG-AAT-GA | |||
|- align="center" | |||
| Disease Reverse Primer||CCC-TTA-TCA-GGG-GGA-CAT-TT | |||
|- | |||
|} | |||
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<!-- INSTRUCTIONS: Write a short summary of the results of your primer test. Underneath your summary, include a screen capture of the results web page. You may crop the image so that it only includes the relevant information. --> | <!-- INSTRUCTIONS: Write a short summary of the results of your primer test. Underneath your summary, include a screen capture of the results web page. You may crop the image so that it only includes the relevant information. --> | ||
The image below shows the UCSC In-Silico PCR website results for the non-disease forward primer and non-disease reverse primer. | |||
[[Image:C1.png]] | |||
The image below shows the UCSC In-Silico PCR website results for the disease specific primers. The results show that there is "no matches." | |||
[[Image:C2.png]] | |||
Latest revision as of 22:34, 11 November 2014
BME 100 Fall 2014 | 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-UPProcedureSmart Phone Camera Settings
Put the glass slide in the sharps container once all the possible white circle have been used. Bonus: Images of Setup Above is an image of the basic setup without the black box over it. Below is an image of the setup with the black box over it but without closing the final side so that we could take the picture.
Data AnalysisRepresentative Images of Negative and Positive Samples Negative Sample Positive Sample
Image J Values for All Calibrator Samples
Image J Data
PCR Results Summary
Observed results
Conclusions
SNP Information & Primer DesignBackground: About the Disease SNP
The image below shows the UCSC In-Silico PCR website results for the non-disease forward primer and non-disease reverse primer. The image below shows the UCSC In-Silico PCR website results for the disease specific primers. The results show that there is "no matches."
|