BME103:T130 Group 1

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(Research and Development)
(OUR TEAM)
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| [[Image:BME103student.jpg|100px|thumb|Name: Bryce Hicok<br>Open PCR machine engineer]]
| [[Image:BME103student.jpg|100px|thumb|Name: Bryce Hicok<br>Open PCR machine engineer]]
| [[Image:BME103student.jpg|100px|thumb|Name: Jesus Ibarra<br>Experimental protocol planner]]
| [[Image:BME103student.jpg|100px|thumb|Name: Jesus Ibarra<br>Experimental protocol planner]]
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| [[Image:BME103student.jpg|100px|thumb|Name: Emma Maiorella<br>R&D scientist]]
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| [[Image:Greater flamingo1.jpg|100px|thumb|Name: Emma Maiorella<br>R&D scientist]]
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Revision as of 21:34, 14 November 2012

BME 103 Fall 2012 Home
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Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
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Contents

OUR TEAM

Name: Tianzhu ZhuExperimental protocol planner
Name: Tianzhu Zhu
Experimental protocol planner
Name: Wyatt HansenOpen PCR machine engineer
Name: Wyatt Hansen
Open PCR machine engineer
Name: Bryce HicokOpen PCR machine engineer
Name: Bryce Hicok
Open PCR machine engineer
Name: Jesus IbarraExperimental protocol planner
Name: Jesus Ibarra
Experimental protocol planner
Name: Emma MaiorellaR&D scientist
Name: Emma Maiorella
R&D scientist

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design

The OpenPCR runs from your computer and connects through your USB port. You can add and delete steps, edit temperatures, and create thermocycler protocols from your computer.


Experimenting With the Connections

- When the PCB board of LCD is unlplugged from the Circuit Board, the LCD goes off.

- When the white wire connecting the circuit board to the heat plate, the LCD showed an incorrect reading of the temperature.


Test Run

Our First practice run was conducted on October 25th, 2012. Our experience with the open PCR went smoothly. The test run took approximately one hour and forty five minutes. It was easy to set up and begin a test run. The only trouble we ran into was when adjusting the heat lid it was hard to tell when the lid was tightened down enough and it if very easy to tighten it too much and squish the test tubes in the PCR.




Protocols

Polymerase Chain Reaction

Polymerase Chain Reaction is the process of rapid duplication of a strand of DNA. The process first requires a DNA template, which contains the strand of DNA that is intended to be copied multiple times. The template strand is then been heated in order to separate the double stranded DNA and DNA Polymerase is added in order to create a strand of DNA that is complimentary to the original intended DNA strand, which creates a primer. Then the temperature is lowered so that the nucleotides will bind together in complementary to the primers in order to create a copy of the targeted DNA strand. The heating and cooling process will be repeated over a period of approximately 2 hours in order to create a large amount of DNA strand copies.

Steps to amplify DNA

   1.) Collect a blood sample from the patient.
   2.) Use chemicals and centrifuge to separate the DNA materials from the rest of the blood 
      components.
   3.) Apply heat (95 degree Celsius) for 3 minutes to the DNA strand to separate the double stranded DNA into 2 single strands.
   4.) In stage two, there are 35 cycles which consists of 95 degree Celsius for 30 seconds. 
   5.) Add primers and cool (57 degree Celsius, then 72 degree Celsius) the sample to allow the primers to bind to the separated original DNA strands. 
   6.) Then reheat the samples again to separated the resulting replicated strands.
   7.) Add primers and cool the samples again.
   8.) Repeat the process of heating and cooling in order for the primers to bind to all the
      separated DNA strands. 
   9.) Over the course of 2 hours or so, there will be a large amount of duplicated DNA strands.

For a 25μL reaction volume

    components                         volume       Final conc
       GoTaq® Colorless Master Mix, 2X          12.5µl             1X
       upstream primer,10µM                     0.25–2.5µl         0.1–1.0µM
       downstream primer, 10µM                  0.25–2.5µl         0.1–1.0µM
       DNA template                             1–5µl              <250ng
       Nuclease-Free Water to                   25µl               N.A

For a 50μL reaction volume

    components                         volume       Final conc
       GoTaq® Colorless Master Mix, 2X          25.0µl             1X
       upstream primer,10µM                     0.50-5.0µl         0.1–1.0µM
       downstream primer, 10µM                  0.50–5.0µl         0.1–1.0µM
       DNA template                             1–5µl              <250ng
       Nuclease-Free Water to                   50µl               N.A


For a 100μL reaction volume

   components                         volume       Final conc
       GoTaq® Colorless Master Mix, 2X          50.0µl             1X
       upstream primer,10µM                     1.00–10.0µl         0.1–1.0µM
       downstream primer, 10µM                  1.00–10.0µl         0.1–1.0µM
       DNA template                             1–5µl              <250ng
       Nuclease-Free Water to                   100µl               N.A

all credit goes to: http://www.promega.com/resources/protocols/product-information-sheets/g/gotaq-colorless-master-mix-m714-protocol/




Flourimeter Measurements

Procedures for Fluorimeter Assembly:

   1.)Place glass slide down on fluorimeter. 
   2.)Place a drop of water on the slide then another close by to form one large drop. 
   3.)Turn on light so that it may pass through water drop.  
   4.)Move the system into the black box. 
   5.)Place smart phone in stand. 
   6.)Take picture and record image. 




Research and Development

Specific Cancer Marker Detection - The Underlying Technology

There are many factors that contribute to a PCR reaction. First, a sample of DNA from a patient must be extracted. This sample is called template DNA. During the reaction, primers, or artifically synthesized bits of DNA, bind to the target sequence if it is present in the DNA. The enzyme taq polymerase's job is to regenerate the DNA strand that was melted away. Magnesium chloride (MgCl2) binds to the taq polymerase protein as a cofactor to help it function properly. In addition, there are dNTP's floating around in the solution of DNA. These deoxynucleotidetriphosphates are the bases (A,T,C,G) ready to be bound by the taq polymerase enzyme.

During a PCR reaction, the solution of DNA is first heated to a temperature of 95 degrees Celsius to essentially melt the hydrogen bonds between the bases on the double strand of DNA. Next, the solution is cooled down to 57 degrees Celsius so the primers can bind to the target sequence of the DNA if it is present. Finally, the solution is heated up to 72 degrees Celsius, where polymerization occurs. This process of heating and cooling is repeated for a total of 34 cycles. This will ensure that the DNA has been amplified enough so it is able to be seen when a flourescent solution is added.

Since the primers will not bind to the DNA if the target sequence is not present, a non-cancer patient will produce a negative result. This is because instead of having millions of double stranded DNA in the solution, the solution will only contain single strands.

The specific cancer-associated gene sequence being analyzed is the r17879961 SNP. The mutation is present at position number 29,121,087 in the DNA. The "ATT" sequence in a non-cancer human is replaced by the "ACT" sequence. Therefore, during a PCR reaction, primers will bind to the sequence, "ACT", since it is the cancer gene.


Description of image

This image shows what happens to the DNA strand each time it replicates. First, the bonds are melted and separated. Then, primers attach themselves to each end of the DNA strand. Next, taq polymerase adds the appropriate bases to create a new piece of double-stranded DNA. Finally, this process is repeated for approximately 30 cycles.

Image borrowed from: [1]




Results

ImageJ Software Processing

Patient ID or Background Area INTDEN RAWINTDEN
Patient 101 78380 45.989 3604632
Background 101 78380 1.215 95224
Patient 102 82228 44.098 3626088
Background 102 82228 .896 73708
Patient 103 80244 65.783 5278725
Background 103 80244 .777 62338
Patient 104 90368 37.274 3368381
Background 104 90368 2.346 211974
Patient 105 99512 181.282 18039714
Background 105 99512 1.786 177715
Patient 106 111476 191.028 21295059
Background 106 111476 .912 101693
Patient 107 88468 224.745 19882762
Background 107 88468 2.026 179269
Patient 108 68088 .953 64894
Background 108 68088 .953 64894
Patient 109 109904 195.414 21476774
Background 109 109904 1.335 146762
Patient 110 69680 95.314 6641513
Background 110 69680 1.981 138023


Sample Integrated Density DNA μg/mL Conclusion
PCR: Negative Control E6 F6 G6
PCR: Positive Control E7 F7 G7
PCR: Patient 1 ID 48199, rep 1 E8 F8 G8
PCR: Patient 1 ID 48199, rep 2 E9 F9 G9
PCR: Patient 1 ID 48199, rep 3 E10 F10 G10
PCR: Patient 2 ID 15130, rep 1 E11 F11 G11
PCR: Patient 2 ID 15130, rep 2 E12 F12 G12
PCR: Patient 2 ID 15130, rep 3 E13 F13 G13


KEY

  • Sample =
  • Integrated Density =
  • DNA μg/mL =
  • Conclusion =


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