BME103 s2013:T900 Group6 L3: Difference between revisions
(22 intermediate revisions by 4 users not shown) | |||
Line 14: | Line 14: | ||
|- valign="top" | |- valign="top" | ||
| [[Image:labtwo.jpg|100px|thumb|Name: Cyril Wassef - Design Strategy-Machine/Device Engineer<br>]] | | [[Image:labtwo.jpg|100px|thumb|Name: Cyril Wassef - Design Strategy-Machine/Device Engineer<br>]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: | | [[Image:BME103student.jpg|100px|thumb|Name: Israel Brewer<br>Research & Development]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: | | [[Image:BME103student.jpg|100px|thumb|Name: Manny Casildo - Research and Development - R&D Scientist <br>]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: | | [[Image:BME103student.jpg|100px|thumb|Name: Dale Caagbay <br>Role(s) Protocol and R&D]] | ||
| [[Image:BME103student.jpg|100px|thumb|Name: Student<br>Role(s)]] | | [[Image:BME103student.jpg|100px|thumb|Name: Student<br>Role(s)]] | ||
|} | |} | ||
Line 128: | Line 128: | ||
<!-- If your team decided to change any of the machinery/ devices, summarize the new features here and delete the '''We chose keep the devices the same as the original system''' section. --> | <!-- If your team decided to change any of the machinery/ devices, summarize the new features here and delete the '''We chose keep the devices the same as the original system''' section. --> | ||
'''We chose to include these new features''' | '''We chose to include these new features''' | ||
* We changed the side panels of the PCR machine to gorilla glass, the bottom to plexiglass, and the front to just glass. This will ensure that the machine won't set on fire. | * We changed the side panels of the PCR machine to gorilla glass, the bottom to plexiglass, and the front to just glass. This will ensure that the machine won't set on fire. This will also increase visibility of what is occurring during the machine from the glass. This will lead to more opportunities to see errors and improve them as the group concludes as necessary. | ||
'''INSTRUCTIONS''' <br> | '''INSTRUCTIONS''' <br> | ||
* First, the machine should be set up in such a way that the machine can be opened easily and accessed. | |||
* Disassemble the machine with a screwdriver on the sides first | |||
* Take each side and assemble each with appropriate glue to the sides; attaching to each other | |||
* Let the machine sit and dry; approximately 2 hours at least optimally. | |||
*'''Follow these instructions to assemble the PCR Machine''' | |||
<!-- Changing the machine will require new instructions for using the machine. Write a short step-by-step list of instructions on how to set up and use the new machine. The instructions must be specific to your new design. --> | <!-- Changing the machine will require new instructions for using the machine. Write a short step-by-step list of instructions on how to set up and use the new machine. The instructions must be specific to your new design. --> | ||
<!-- If your team decided not to change any of the machinery/ devices, write a short step-by-step list of instructions on how to set up and use each machine. You may want to consider modifying the instructions to improve ease of use. --> | <!-- If your team decided not to change any of the machinery/ devices, write a short step-by-step list of instructions on how to set up and use each machine. You may want to consider modifying the instructions to improve ease of use. --> | ||
Line 142: | Line 147: | ||
'''DESIGN''' | '''DESIGN''' | ||
*We decided to change the materials of the PCR machine in order for it to be a safer machine. | |||
<!-- If your team decided to change the PCR and/or the Fluorimeter imaging protocols, summarize the new approaches/ features here and delete the '''We chose keep the protocols the same as the original system''' section. --> | <!-- If your team decided to change the PCR and/or the Fluorimeter imaging protocols, summarize the new approaches/ features here and delete the '''We chose keep the protocols the same as the original system''' section. --> | ||
Line 154: | Line 154: | ||
Supplied in Kit | Supplied in Kit | ||
* | *MgCl2 | ||
* | *dNTP | ||
* | *Reaction Buffer | ||
*DNA polymerase | |||
* | |||
User | |||
*Sample DNA | |||
*Primers | |||
*SYBR Green Dye | |||
Line 170: | Line 172: | ||
* Feature 1 - The PCR machine effectively amplifies the DNA with little error. | * Feature 1 - The PCR machine effectively amplifies the DNA with little error. | ||
* Feature 2 - The software used in the PCR testing is very easy to use. | * Feature 2 - The software used in the PCR testing is very easy to use. | ||
* Feature 3 - The | * Feature 3 - The fluorimeter gave us a good idea whether a sample was positive or negative | ||
'''DNA Measurement and Analysis Protocol''' | |||
<!-- Create a step-by-step procedure for measuring DNA amplification in the PCR reactions. Your instructions should include everything from diluting the samples in SYBR Green, to placing the drops onto the fluorimeter (if your group is using the fluorimeter), to collecting and processing images in Image J. Don't forget to provide instructions on how to set up the calf thymus DNA samples for calibration, and how to convert INTDEN values into concentrations.---> | <!-- Create a step-by-step procedure for measuring DNA amplification in the PCR reactions. Your instructions should include everything from diluting the samples in SYBR Green, to placing the drops onto the fluorimeter (if your group is using the fluorimeter), to collecting and processing images in Image J. Don't forget to provide instructions on how to set up the calf thymus DNA samples for calibration, and how to convert INTDEN values into concentrations.---> | ||
# Step 1: Gather materials and safety equipment | # Step 1: Gather materials and safety equipment | ||
# Step 2: Put | # Step 2: Put fluorimeter in dark box, keep one hatch open for ease of access | ||
# Step 3: Level the phone camera lens with | # Step 3: Level the phone camera lens with fluorimeter | ||
# Step 4: Calibrate | # Step 4: Calibrate fluorimeter with water | ||
# Step 5: Dilute samples with SYBR | # Step 5: Dilute samples with SYBR Green (80ug) | ||
# Step 6: Run | # Step 6: Place 80ug of solution onto fluorimeter slide (make sure blue light runs through drop) | ||
# Step | # Step 7: Run fluorimeter again | ||
# Step | # Step 8: Take picture and repeat with the different samples | ||
# Step | # Step 9: Open image J | ||
# Step | # Step 10: Split picture into three channels and choose green | ||
# Step 10: Create a circle around the droplet in picture | |||
# Step 11: FInd the aveintdent for each droplet by clicking analyze, then measure | |||
{| {{table}} width=700 | |||
|- | |||
| Calf Thymus DNA solution concentration (microg/mL) || Volume of the 2X DNA solution (uL) || Volume of the SYBR GREEN I Dye Solution (uL) || Final DNA concentration in PicoGreen Assay (ng/mL) | |||
|- | |||
| 0 || 80 || 80 || blank | |||
|- | |||
| .25 || 80 || 80 || .125 | |||
|- | |||
| .5 || 80 || 80 || .25 | |||
|- | |||
| 1 || 80 || 80 || .5 | |||
|- | |||
| 2 || 80 || 80 || 1 | |||
|- | |||
| 5 || 80 || 80 || 2.5 | |||
|} | |||
'''Smart Phone Camera Settings'''<br> | |||
* ''Iphone 5'' | |||
** Flash: None | |||
** ISO setting: NA | |||
** White Balance: NA | |||
** Exposure: NA | |||
** Saturation: NA | |||
** Contrast: NA | |||
<br><br> | <br><br> | ||
Line 191: | Line 222: | ||
'''BACKGROUND''' | '''BACKGROUND''' | ||
* In summary the CHEK2 gene, short for Checkpoint Kinase 2, encodes the protein that inhibits the CSC25C phosphatase when activated. In other words, it helps to slow down cell growth and division. This particular phosphatase helps to "counter" the tumor cells and bring in a better chance of survival for damaged and affected cells. It slows down the tumor cell's rapid growth rate and allow medications to work on affect areas. Associating with this gene, SNP, short for 'Single Nucleotide Polymorphism', is clinically significant in that it is a pathogenic substance. In clarification, it shows if cancer is evident in cells or not. Because of it's pathogenic properties, it flips the function of CHEK2 in reverse and promotes cancer cell growth. | |||
In the worksheet that we've filled out, we found that the sequence for CHEK2 is ATT. If it is altered in anyway, we can come to conclusions that there may be the presence of a carcinogen in cells. We can verify this by the mutation from ATT to ACT. | |||
Source: http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=17879961 | |||
'''DESIGN''' | '''DESIGN''' | ||
* The PCR machine will include nucleotides that will bind to certain base pairs and promote cancer cell growth. This is very helpful in a visual sense, obviously if the patient has cancer then cancer cells will grow to promote visibility of the cells and accuracy of the PCR machine. | |||
'''Primers for PCR'''<br> | '''Primers for PCR'''<br> | ||
<!-- If your team decided to only amplify cancer-associated DNA, list the "Cancer allele forward primer" sequence and the "Cancer allele reverse primer" sequence. Include a paragraph that explains why a disease allele will give a PCR product and the non-disease allele will not.--> | <!-- If your team decided to only amplify cancer-associated DNA, list the "Cancer allele forward primer" sequence and the "Cancer allele reverse primer" sequence. Include a paragraph that explains why a disease allele will give a PCR product and the non-disease allele will not.--> | ||
* The primers that were selected was: Forward primer: Reverse Primer: | |||
* A disease allele will give a PCR product because the primers that were selected are only going to match the DNA sequence that is cancerous. When the primer binds to a cancerous allele it will then start to grow thus visible if cancerous. However there will not be a PCR product if there is a non disease allele for the same reason. When the primer and the DNA are put together there will not be any binding because the nucleotides will not match correctly onto the DNA thus negating any replication and any visibility or product. | |||
* | |||
Latest revision as of 09:16, 16 April 2013
BME 103 Spring 2013 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAMLAB 3 WRITE-UPOriginal System: PCR ResultsPCR Test Results
* Ave. INTDEN = Average of ImageJ integrated density values from three Fluorimeter images
Bayes Theorem equation: P(A|B) = P(B|A) * P(A) / P(B)
Calculation 3: The probability that the patient will develop cancer, given a cancer DNA sequence.
New System: Design StrategyWe concluded that a good system Must Have:
New System: Machine/ Device EngineeringSYSTEM DESIGN
KEY FEATURES We chose to include these new features
New System: ProtocolsDESIGN
Supplied in Kit
User
We chose keep the protocols the same as the original system
DNA Measurement and Analysis Protocol
New System: Research and DevelopmentBACKGROUND
In the worksheet that we've filled out, we found that the sequence for CHEK2 is ATT. If it is altered in anyway, we can come to conclusions that there may be the presence of a carcinogen in cells. We can verify this by the mutation from ATT to ACT. Source: http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=17879961
|