|Line 62:||Line 62:|
Components of the PCR master mix
Components of the PCR master mix
400μM dATP, 400μM dGTP, 400μM dCTP, 400μM dTTP and 3mM MgCl2, reaction buffer (pH 8.5), nuclease-free water
Revision as of 13:37, 14 November 2012
|BME 103 Fall 2012|| Home |
Lab Write-Up 1
Lab Write-Up 2
Lab Write-Up 3
Course Logistics For Instructors
Wiki Editing Help
LAB 1 WRITE-UP
Initial Machine Testing
When we unplugged part mounting plate from circuit board, the machine had no visible screen and we could not see the the information on the LCD screen.
When we unplugged the white wire that connects the circuit board to the sample holder, the machine's temperature was unable to be monitored and could not be regulated
Our group did our test run on October 24, 2012. During the test run, we ran into no problems with the machine. We were done and found our results in a matter of 1 to 2 hours. The Open PCR machine was connected to one computers to control each temperature of each cycle of PCR during the experimental.
Polymerase Chain Reaction
How PCR Works:
Step 1: Denaturation by Heat: Initially, heat separates a DNA strand into two separate strands. This is allowed because the hydrogen bonds holding the DNA together are weak and easily separable when heated.
Step 2: Annealing Primer to Target Sequence: We want to target a sequence specifically and to do that you must use primers. Primers mark the ends of the target sequence. Two primers are included in the PCR one for each of the strands that were just separated during denaturation. The beginning of the DNA target sequence is also marked by the primers that bind (anneal) to the complementary sequence.
Step 3: Extension: After the primers bind to the complementary DNA, the temperature is raised and the enzyme Taq DNA polymerase is used to replicate the strands. The Taq DNA polymerase, active at high temperatures, facilitates the binding and joining of the complementary nucleotides. It synthesizes an identical double-stranded DNA strand. Extension begins at the 3’ end of the primer because Taq DNA polymerase synthesizes exclusively in the 5’ to 3’ direction, so the free nucleotides are only added to the 3’ end of the primer.
How to Amplify a Patient’s DNA sample 1. A PCR Machine is created to amplify a patient’s DNA sample. In order to use a PCR machine, the steps include to first gather the DNA samples, 50 μL each. 2. The frozen DNA samples are separated into tubes and once melted, 8 transfer pipettes were used to add primer. 3. Once the samples are ready, we processed the DNA in the openPCR system.
Fluorimeter assembly procedure: Turn on the excitation light on the fluorimeter. Put a smart phone in the cradle and set it up to take pictures of the slide. Place two drops of water in the middle of the first two rows of the slide using a pipette. Align the drop by moving the slide so the drop is in the middle of the black fiber optic fitting. Cover the fluorimeter with the light box while still being able to use the smart phone to take pictures.
How to open images in Image J- Save the pictures to the phone. Download the pictures onto a computer that has Image J. Open them with Image J by going to add image.
Research and Development
Specific Cancer Marker Detection - The Underlying Technology
Cancer Associated with the gene: Breast and Colorectal cancer with a susceptibility to LiFraumeni Syndrome
Normal Gene Sequence:
Cancer Gene Sequence:
Description--------INTDEN Subtracted Background----------DNA Concentration (micrograms/mL)
Water Blank----------218527------------------------------- 0