BME103:T930 Group 14 l2

From OpenWetWare
Jump to navigationJump to search
BME 103 Fall 2012 Home
People
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Photos
Wiki Editing Help

OUR TEAM

Name: Jake Lindquist
Protocol Planner
Name: Breanna Pratt
Protocol Planner
Name: Kirsten Jefferys
Open PCR Machine Engineer
Name: Ben Alcorn
Open PCR Machine Engineer
Name: Carlos Duarte
Research and Design Scientist
Name: Bryce DeSimmone
Research and Design Scientist

LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.


System Design

What changes are we making to our system? Will have to take another picture with changes before class!


Key Features
Our re-designed system will have the adjusting screw removed, and instead will be fixed at a set level so that the operator will not have to fiddle with adjusting the lid and can simply just open and close the lid with ease without worrying about the height of the heating plate.


Instructions
The instructions for this system are the same as the old system, minus having to adjust the height of the heating plate.




Protocols

Materials

Supplied in the kit Amount
PCR Assembly 1
Fluorimeter 1
Phone Stand 1
Box 1


Supplied by User Amount
Samples 3 samples per subject
Positive & Negative controls 1 each
Calibrator (Calif.../water blank) 1 each
Enzyme/Primer mix Enough for all the samples
Test tubes 1 for each sample, and control
Pippettes 1 for each sample, control, and calibration solution


PCR Protocol

  • Procedure:
    • Preparing the DNA samples
  1. Obtain 6 DNA samples, 2 subjects with 3 samples from each subject and clearly label each test tube. Also include 2 extra tubes, a positive and a negative control
  2. Add 50 μL of the PCR reaction mixture to each 50 μL sample of patient DNA. The PCR reaction mix is composed of:
    1. 0.2 μL Template DNA (20 ng)
    2. 1.0 μL 10 μM forward primer
    3. 1.0 μL 10 μM reverse primer
    4. 50.0 μL GoTaq master mix
      1. 2X Colorless GoTaq® Reaction Buffer( pH 8.5)
      2. 400μM dATP
      3. 400μM dGTP
      4. 400μM dCTP
      5. 400μM dTTP
      6. 3mM MgCl2.
    5. 47.8 μL dH2O (A total volume of 100.0 μL)
  3. The eight prepared samples should be placed into a refrigerator until they are placed into the Open PCR Machine.
    • Setting up the Open PCR Machine
  1. The Open PCR Machine must be plugged in and connected to a computer through a USB port and cable.
  2. The PCR machine holds 16 samples, only 8 of the wells will be used in this situation. Place the wells horizontally to the lid hinge, in the inner well rows.
  3. Close the lid and ensure that it snaps down completely
  4. Once the machine is turned on and plugged into the computer, with software already downloaded, the test should be programed as follows
    1. 30 cycles must be run of the following pattern
      1. 90ºC for 30 seconds
      2. 57ºC for 30 seconds
      3. 72ºC for 30 seconds



DNA Measurement Protocol

Research and Development

Background on Disease Markers

ALZHEIMER'S DISEASE (AD)

As it turns out, Alzheimer's Disease is a uniquely diverse disease, as it has many different genetic mutations that can cause early-onset Alzheimer's. A brief background before we start. Early-onset AD is the least common form of AD, as it only occurs in 5% of individuals who have the disease, but it is the only type of AD that comes almost completely from inherited genetic traits. The problem comes in when the new gene sequence causes a change in a protein made, which generates harmful amyloid plaques (the driving force of the disease). Late-onset AD occurs in the other 95% and is a combination of lifestyle, genetic, and environmental factors.

Most of info found on: (http://www.stanford.edu/class/gene210/files/projects/Gen210AlzheimersDisease.pdf)


Primer Design

ALZHEIMER'S DISEASE (AD)

Because there are many different variations of genetic early-onset AD that can occur, we chose to focus on the sequence rs17517621, which causes a G to change to an A. AAATCTTTTTG[G/A]CAAATTTG is the specific primer sequence that we located for this disease. Following the DNA strand to the left, the specific primer for this type of genetic AD variation was found. According to Dr. Haynes, only 150 BP to the left are needed, so we only went 150 BP to help increase the speed of the PCR. The DNA primer sequence is GACAATTGCTAAGTGTAACA (http://www.ncbi.nlm.nih.gov/snp?term=17517621), which can be used, as discussed before, to help identify DNA with this genetic variation present. And the reverse would be CTGTTAACGATTCACATTGT.

Forward Primer:GACAATTGCTAAGTGAACA Reverse Primer:ACAAGTGAATCGTTAACAG

Other common variances of AD occur in rs429358 and rs7412 (which involve changes in C and T), but the primer and sequence is only needed for rs17517621. As discussed in the last lab, a diseased allele will give a positive result in the PCR because only this specific primer can bind to that specific DNA sequence. So if the disease is present, the primer will bind and replicate the DNA exponentially, resulting in a positive. If the disease is not present, on the other hand, the primer will have no chance to bind, thus giving a negative result.


Illustration