BME100 f2014:Group23 L5: Difference between revisions
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{| style="wikitable" width="700px" | {| style="wikitable" width="700px" | ||
|- valign="top" | |- valign="top" | ||
| [[Image: | | [[Image:1-.jpeg|100px|thumb|Name: Cesar Marin]] | ||
| [[Image: | | [[Image:IMG 0082 converted.jpg|100px|thumb|Name: Brady Dennison]] | ||
| [[Image: | | [[Image: IMG_3058.pdf ( 79 Kb ) |100px|thumb|Name: Kassandra Flores]] | ||
| [[Image: | | [[Image:024 IMG 0062.JPG|100px|thumb|Name: Danielle Beach]] | ||
| [[Image: | | [[Image:Tedsphoto10304982_10152025365627046_4574490114725615102_n_(2).jpg|100px|thumb|Name: Theodore Kyriacou]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: | | [[Image:BME103student.jpg|100px|thumb|Name: Joshua Kahn]] | ||
|} | |} | ||
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'''Smart Phone Camera Settings'''<br> | '''Smart Phone Camera Settings'''<br> | ||
<!-- The type of smart phone you used and how you adjusted the camera settings, if applicable. If you used more than one phone, make an additional list. --> | <!-- The type of smart phone you used and how you adjusted the camera settings, if applicable. If you used more than one phone, make an additional list. --> | ||
* Type of Smartphone: | * Type of Smartphone: Samsung Galaxy Note 3 | ||
** Flash: | ** Flash: None | ||
** ISO setting: | ** ISO setting: 800 | ||
** White Balance: | ** White Balance: Auto | ||
** Exposure: | ** Exposure: Highest Setting | ||
** Saturation: | ** Saturation: Highest Setting | ||
** Contrast: | ** Contrast: Highest Setting | ||
'''Calibration'''<br> | '''Calibration'''<br> | ||
<!-- INSTRUCTIONS: In the space below, briefly describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points. --> | <!-- INSTRUCTIONS: In the space below, briefly describe how to set up your camera in front of the fluorimeter. Add a PHOTO of this set-up for bonus points. --> | ||
The smartphone was placed in the cradle with the camera app on. The cradle was then moved so that the phone was 12 cm away from from where the drop would be measured. Pieces of bubble wrap were placed in between the phone and the cradle so that the phone was level with the drop. | |||
<!-- INSTRUCTIONS: Type the distance between your phone cradle and the drop after the equal sign. --> | <!-- INSTRUCTIONS: Type the distance between your phone cradle and the drop after the equal sign. --> | ||
* Distance between the smart phone cradle and drop = | * Distance between the smart phone cradle and drop = 12 cm | ||
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{| {{table}} width=700 | {| {{table}} width=700 | ||
|- | |- | ||
| | | Initial Concentration of 2X Calf Thymus DNA Solution (µg/mL) || Volume of 2X DNA Solution (µL) || Volume of SYBR Green I Dye Solution (µL) || Final DNA Concentration in SYBR Green I Solution (µg/mL) | ||
|- | |||
| 5 || 80 || 80 || 2.5 | |||
|- | |||
| 2 || 80 || 80 || 1 | |||
|- | |- | ||
| | | 1 || 80 || 80 || 0.5 | ||
|- | |- | ||
| | | 0.5 || 80 || 80 || 0.25 | ||
|- | |||
| 0.25 || 80 || 80 || 0.125 | |||
|- | |||
| 0 || 80 || 80 || 0 | |||
|} | |} | ||
'''Solutions Used for Measuring PCR Products''' | |||
{| {{table}} width=700 | |||
|- | |||
| PCR Product Tube Label || Volume of the Diluted PCR Product solution(μL) || Volume of SYBR Green I Dye Solution (µL) || Dilution 1 || Dilution 2 || Total Dilution Simplified Fraction | |||
|- | |||
| G23 + || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 - || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 1-1 || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 1-2 || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 1-3 || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 2-1 || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 2-2 || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|- | |||
| G23 2-3 || 80 || 80 || 0.166 || 0.5 || 0.0833 | |||
|} | |||
<!-- Add more rows and cells as needed. --> | <!-- Add more rows and cells as needed. --> | ||
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'''Placing Samples onto the Fluorimeter''' | '''Placing Samples onto the Fluorimeter''' | ||
# '' | # ''Line up fluorimeter light with the section on slide where the sample will be placed (middle of two circles)'' | ||
# '' | # ''Put new tip into micropipette and collect 80uL of SYBR green'' | ||
# '' | # ''Place 80 uL of SYBR green into area from step 1, lined with the light'' | ||
# '' | # ''Discard tip, get new tip and collect 80uL of desired sample'' | ||
# ''Place 80uL of sample on top of SYBR green in the fluorimeter slide'' | |||
# ''Adjust slide on fluorimeter to allow for the narrowest possible stream of light'' | |||
# ''Place camera on a timer'' | |||
# ''Place camera and fluorimeter in a box to block out light, and wait until picture is taken'' | |||
<br> | <br> | ||
<!-- Note: Be sure to delete the instruction text in brackets: ''[ ]'' --> | <!-- Note: Be sure to delete the instruction text in brackets: ''[ ]'' --> | ||
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<!-- 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. --> | ||
Sample With No DNA | |||
[[Image:No DNA.png]] | |||
Sample With DNA | |||
[[Image:DNAA.png]] | |||
'''Image J Values for All Calibrator Samples''' | '''Image J Values for All Calibrator Samples''' | ||
Line 80: | Line 120: | ||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Final DNA concentration in SYBR Green I solution (µg/mL)''' | |||
| align="center" style="background:#f0f0f0;"|'''Area''' | |||
| align="center" style="background:#f0f0f0;"|'''Mean Pixel Value''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN of the Drop''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN of the Background''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN Drop - Background''' | |||
|- | |||
| 0#1||117281||62.946||7382324||120175||7262149 | |||
|- | |||
| 0#2||95944||55.867||5360083||82761||5277322 | |||
|- | |||
| 0#3||102480||56.232||5762676||122017||5640659 | |||
|- | |||
| 0.125#1||200967||58.129||11682080||230881||11451199 | |||
|- | |||
| 0.125#2||209397||61.156||12805862||316028||12489834 | |||
|- | |||
| 0.125#3||220281||59.243||13050128||315441||12734687 | |||
|- | |||
| 0.25#1||148276||105.684||15670365||166860||15503505 | |||
|- | |||
| 0.25#2||143862||104.852||15084237||168573||14915664 | |||
|- | |||
| 0.25#3||148848||104.226||15513804||176779||15337025 | |||
|- | |||
| 0.5#1||63004||121.832||7675924||84689||7591235 | |||
|- | |||
| 0.5#2||53899||121.386||6542599||67056||6475543 | |||
|- | |||
| 0.5#3||63250||120.075||7594743||71477||7523266 | |||
|- | |||
| 1#1||101788||159.563||16241620||111100||16130520 | |||
|- | |||
| 1#2||107815||154.656||16674224||121570||16552654 | |||
|- | |||
| 1#3||106365||155.667||16557507||116040||16441467 | |||
|- | |||
| 2.5#1||52043||224.116||11663647||272607||11391040 | |||
|- | |||
| 2.5#2||96387||201.455||19417686||339679||19078007 | |||
|- | |||
| 2.5#3||93996||203.554||19133220||489494||18643726 | |||
|} | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''PCR Product Tube Label''' | |||
| align="center" style="background:#f0f0f0;"|'''Area''' | |||
| align="center" style="background:#f0f0f0;"|'''Mean Pixel Value''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN of the Drop''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN of the Background''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN Drop - Background''' | |||
|- | |||
| G23 + #1||81288||211.873||17222739||519304||16703435 | |||
|- | |||
| G23 + #2||83298||209.743||17471210||477364||16993846 | |||
|- | |||
| G23 + #3||84916||206.982||17576093||380353||17195740 | |||
|- | |||
| G23 - #1||63262||47.935||3032495||114182||2918313 | |||
|- | |||
| G23 - #2||65364||52.551||3434964||126152||3308812 | |||
|- | |||
| G23 - #3||73048||53.772||3927959||184799||3743160 | |||
|- | |||
| G23 1-1 #1||63236||217.135||13730756||291772||13438984 | |||
|- | |||
| G23 1-1 #2||69676||214.228||14926555||274659||14651896 | |||
|- | |||
| G23 1-1 #3||72935||209.691||15293786||484533||14809253 | |||
|- | |||
| G23 1-2 #1||68930||223.918||15434635||748054||14686581 | |||
|- | |||
| G23 1-2 #2||83892||214.71||18012462||542818||17469644 | |||
|- | |||
| G23 1-2 #3||78844||213.253||16813706||552361||16261345 | |||
|- | |||
| G23 1-3 #1||79201||218.261||17286505||653016||16633489 | |||
|- | |||
| G23 1-3 #2||91457||222.257||20326925||1007852||19319073 | |||
|- | |||
| G23 1-3 #3||77372||225.738||17465838||697357||16768481 | |||
|- | |||
| G23 2-1 #1||86508||221.876||19194089||849243||18344846 | |||
|- | |||
| G23 2-1 #2||79512||230.236||18306536||717249||17589287 | |||
|- | |||
| G23 2-1 #3||84287||228.699||19276328||708500||18567828 | |||
|- | |||
| G23 2-2 #1||77942||224.541||17501204||264074||17237130 | |||
|- | |||
| G23 2-2 #2||66272||229.613||15216881||260396||14956485 | |||
|- | |||
| G23 2-2 #3||69675||229.864||16015757||250409||15765348 | |||
|- | |||
| G23 2-3 #1||46398||225.599||10467352||271372||10195980 | |||
|- | |||
| G23 2-3 #2||39267||223.424||8773180||218858||8554322 | |||
|- | |||
| G23 2-3 #3||54020||228.365||12336293||436545||11899748 | |||
|} | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''Final DNA concentration in SYBR Green I solution (µg/mL)''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN DROP - BACKGROUND''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| ||1||2||3||Mean||Standard Deviation | |||
|- | |||
| 0||7262149||5277322||5640659||6060043.333||1056786.151 | |||
|- | |||
| 0.125||11451199||12489834||12734687||12225240||681427.0689 | |||
|- | |||
| 0.25||15503505||14915664||15337025||15252064.67||302990.0229 | |||
|- | |||
| 0.5||7591235||6475543||7523266||7196681.333||625448.0938 | |||
|- | |||
| 1||16130520||16552654||16441467||16374880.33||5044544.086 | |||
|- | |||
| 2.5||11391040||19078007||18643726||16370924.33||4318169.285 | |||
|} | |||
{| | |||
| align="center" style="background:#f0f0f0;"|'''PCR Product Tube Label''' | |||
| align="center" style="background:#f0f0f0;"|'''RAWINTDEN DROP - BACKGROUND''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| ||1||2||3||Mean | |||
|- | |||
| G23 +||16703435||16993846||17195740||16964340.33 | |||
|- | |||
| G23 -||2918313||3308812||3743160||3323428.333 | |||
|- | |||
| G23 1-1||13438984||14651896||14809253||14300044.33 | |||
|- | |||
| G23 1-2||14686581||17469644||16261345||16139190 | |||
|- | |||
| G23 1-3||16633489||19319073||16768481||17573681 | |||
|-[[Image:[[Image:Example.jpg]]]] | |||
| G23 2-1||18344846||17589287||18567828||18167320.33 | |||
|- | |||
| G23 2-2||17237130||14956485||15765348||15986321 | |||
|- | |||
| G23 2-3||10195980||8554322||11899748||10216683.33 | |||
|} | |||
'''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:Final_DNA_SYBR.png]] | ||
{| | |||
| align="center" style="background:#f0f0f0;"|'''PCR patient label''' | |||
| align="center" style="background:#f0f0f0;"|'''Mean RAWINTDEN drop-background''' | |||
| align="center" style="background:#f0f0f0;"|'''PCR product concentration''' | |||
| align="center" style="background:#f0f0f0;"|'''Initial PCR product concentration''' | |||
|- | |||
| G23 +||16964340.33 ||2.32 ug/mL || 27.84 ug/mL || | |||
|- | |||
| G23 -||3323428.333|| -2.23 ug/mL || -26.76 ug/mL || | |||
|- | |||
| G23 1-1||14300044.33 ||1.43 ug/mL || 17.16 ug/mL || | |||
|- | |||
| G23 1-2||16139190 || 2.05 ug/mL || 24.6 ug/mL || | |||
|- | |||
| G23 1-3||17573681 || 2.52 ug/mL||30.24 ug/mL || | |||
|- | |||
| G23 2-1|| 18167320.33 || 2.72 ug/mL || 32.64 ug/mL || | |||
|- | |||
| G23 2-2|| 15986321 ||1.99 ug/mL || 23.88 ug/mL || | |||
|- | |||
| G23 2-3|| 10216683.33 || .072 ug/mL || 0.864 ug/mL || | |||
|} | |||
'''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 positive control PCR result was 27.84 μg/mL | ||
* Our negative control PCR result was | * Our negative control PCR result was -26.76 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 1-33849 : All of the images for patient 1 were bright green. The green was mostly at the bottom, with blue at the top. Patient 1 had an average initial PCR product concentration of 24 μg/mL. | ||
* Patient | * Patient 2-19967 : All of the images for patient 2 were also bright green. The green fluorescence was shown all throughout the drop. Patient 2 had an average initial PCR product concentration of 18.869 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 | * Patient 1-33849 : Patient 1 had an average initial PCR product concentration that was 3.84 μg/mL less than that of the product concentration of the positive sample, and 50.76 μg/mL greater than that the product concentration of the negative sample. Since Patient 1 had a concentration closer to the concentration of the positive sample, Patient 1 is positive. All of Patient 1's initial concentrations were closer to the concentration of the positive sample than the negative sample. | ||
* Patient | * Patient 2-19967 : Patient 2 had an average initial PCR product concentration that was 8.9712 μg/mL less than that of the product concentration of the positive sample, and 45.6288 μg/mL greater than that the product concentration of the negative sample. Since Patient 2 had a concentration closer to the concentration of the positive sample, Patient 2 is positive. All of Patient 2's initial concentrations were closer to the concentration of the positive sample than the negative sample. Positive initial concentrations suggest that the patient is positive, while negative initial concentrations suggest that the patient is negative. | ||
Line 110: | Line 319: | ||
'''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. --> | ||
A nucleotide is the basic building block of DNA and other nucleic acids. the four main nucleotides are A,C,T,and G. A polymorphism is a common change in DNA sequences that appears in the phenotypes of different groups of population. The SNP rs16991654 variation is found in homosapiens. This variation is found in the 21:34370656 chromosome. The clinical significance is this SNP is that it is pathogenic. This SNP is associated with the KCNE2 gene. This SNP is linked to the disease of congenital long QT syndromes. KCNE2 stands for potassium voltage-gated channel, Isk-related family, member 2. The molecular function of this gene includes regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. An allele in an alternative to a gene sequence that are done through mutation of a nucleotide base pair. The disease-associated allele contains the sequence CTC. The numerical position of the SNP is 34,370,656. The non-disease forward primer is catggtgatgattggaatgt. The numerical position exactly 200 bases to the right of the disease SNP is 3,430,856. The non-disease reverse primer is cccttatcagggggacattt. The diseases forward primer is catggtgatgattggaatgc. The disease reverse primer is cccttatcagggggacattt. | |||
Line 115: | Line 327: | ||
<!-- 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. --> | ||
When the non-disease forward primer was tested, it was found in the database. This is because a part of the DNA that was selected did not contain the sequence that causes the disease. The disease specific primers did not exist because one nucleotide was changed in the DNA sequence. While this may cause the disease, it affects the entire sequence, it does not exist since it did not change by a group of three nucleotides, or an entire codon. | |||
Non-Disease Forward Primer Results | |||
[[Image:Part a.png]] | |||
Disease Specific Primers Results | |||
[[Image:Part b.png]] | |||
Latest revision as of 10:25, 19 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
The smartphone was placed in the cradle with the camera app on. The cradle was then moved so that the phone was 12 cm away from from where the drop would be measured. Pieces of bubble wrap were placed in between the phone and the cradle so that the phone was level with the drop.
Solutions Used for Measuring PCR Products
Placing Samples onto the Fluorimeter
Data AnalysisRepresentative Images of Negative and Positive Samples Sample With No DNA Sample With DNA Image J Values for All Calibrator Samples
PCR Results Summary
Observed results
Conclusions
SNP Information & Primer DesignBackground: About the Disease SNP A nucleotide is the basic building block of DNA and other nucleic acids. the four main nucleotides are A,C,T,and G. A polymorphism is a common change in DNA sequences that appears in the phenotypes of different groups of population. The SNP rs16991654 variation is found in homosapiens. This variation is found in the 21:34370656 chromosome. The clinical significance is this SNP is that it is pathogenic. This SNP is associated with the KCNE2 gene. This SNP is linked to the disease of congenital long QT syndromes. KCNE2 stands for potassium voltage-gated channel, Isk-related family, member 2. The molecular function of this gene includes regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. An allele in an alternative to a gene sequence that are done through mutation of a nucleotide base pair. The disease-associated allele contains the sequence CTC. The numerical position of the SNP is 34,370,656. The non-disease forward primer is catggtgatgattggaatgt. The numerical position exactly 200 bases to the right of the disease SNP is 3,430,856. The non-disease reverse primer is cccttatcagggggacattt. The diseases forward primer is catggtgatgattggaatgc. The disease reverse primer is cccttatcagggggacattt.
Primer Design and Testing When the non-disease forward primer was tested, it was found in the database. This is because a part of the DNA that was selected did not contain the sequence that causes the disease. The disease specific primers did not exist because one nucleotide was changed in the DNA sequence. While this may cause the disease, it affects the entire sequence, it does not exist since it did not change by a group of three nucleotides, or an entire codon. Non-Disease Forward Primer Results Disease Specific Primers Results
|