OUR TEAM

Name: Serena Kaplan Research and Development

Name: Gabe McInnis Open PCR Machine Engineer

Name: Blake Eichler Experimental Protocol Planner

Name: Sierra Morris Experimental Protocol Planner

Name: Zazu Moloi Open PCR Machine Engineer

Name: Katelin Vaughn Research and Development

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

Key Features

Instructions

Protocols

Polymerase Chain Reaction

PCR is a method of amplifying a sample of DNA. PCR alters the temperature and allows the DNA to separate, bind to primers, and catalyze. This results in the amount of DNA doubling after each cycle.
Procedure:
1.) The DNA samples were heated to ninety-five degrees Celsius (95°C) for three (3) minutes to unzip the two single strands.
2.) They were then cooled to fifty-seven degrees Celsius (57°C) and the primers were attached to their matching sequences.
3.) They were then heated back to seventy-two degrees Celsius (72°C) and polymerase extended the DNA strands by attaching the correct free nucleotides in order on the single strands.

GoTaq Mix Components For 100μl reaction volume:
2X GoTaq Colorless Master Mix
10 μM upstream primer
10 μM downstream primer
DNA template
Nuclease-Free Water to

Patient 1
ID 30269
Male, 55 years old

Patient 2
ID 22057
Female, 55 years old

Fluorimeter Setup

1.) The lid was first taken off the the box and one of its sides was unbuttoned in order to create a flap.
2.) The box was the flipped upside down in order to create a dark environment for the camera.
3.) A hydrophobic slide was then inserted into the flourimeter.
4.) Finally, the camera phone was placed in the stand.

Fluorimeter Measurements

Fluorimeter Assembly Procedure
1.) Label transfer pipettes and tubes
2.) Transfer each sample separately into tube containing 400μl of buffer
3.) Take the specifically labeled tube containing SYBR GREEN 1 and place 2 drops on the first 2 centered drops
4.) Place 2 drops of diluted sample on top of the SYBR GREEN 1 drop
5.) Align light through drop
6.) Take pictures using light box
7.) Repeat for each sample.
8.) Run water as BLANK using same procedure

ImageJ Instructions
1.) Open ImageJ
2.) Click ANALYZE tool bar and select SET MEASUREMENTS
3.) Select AREA, MEAN GREY VALUE, and INTEGRATED DENSITY
4.) Upload image to ImageJ
5.) Select IMAGE then COLOR and then SPLIT CHANNELS
6.) Only use green channel
7.) Use OVAL tool and select the entire drop of liquid
8.) Go to ANALYZE and then MEASURE
9.) Drag circle to the background of the image
10.) Record results
11.) Repeat if necessary

Research and Development

Background on Disease Markers

Primer Design

Illustration