BME103:T130 Group 15 l2: Difference between revisions
Line 133: | Line 133: | ||
'''Steps to Amplify DNA Samples'''<br> | '''Steps to Amplify DNA Samples'''<br> | ||
# Collect three replicate DNA samples from two patients. (was provided already) | # Collect three replicate DNA samples from two patients. (was provided already) | ||
# The DNA samples are 50 μL each, get the patient's ID and label the the each tube. | # The DNA samples are 50 μL each, get the patient's ID and label the the each tube. | ||
# PCR reaction mix - Mix contains Taq DNA polymerase, MgCl<sub>2</sub>, dNTP's, forward primer, and reverse primer. | # PCR reaction mix - Mix contains Taq DNA polymerase, MgCl<sub>2</sub>, dNTP's, forward primer, and reverse primer. | ||
Line 147: | Line 141: | ||
# The 8 tubes of mixtures will then go through the cycles in the PCR system. | # The 8 tubes of mixtures will then go through the cycles in the PCR system. | ||
#* During each step of the thermal cycling, the DNA is unzipped and heated to 95°C to break the H-bonds between the 2 strands. This exposes the part we want in this lab experiment. Then, the primer binds to the trage we want without cancer marker, this primer won't bind. Next, the temperature will be dropped to 57°C in order to bind the primer. Later, it is heat it back up to 72°C with the Taq to reform and duplicate DNA strands. Finally, this thermal cycling is repeated for amplification and add dye that binds specifically to DNA for detection. | #* During each step of the thermal cycling, the DNA is unzipped and heated to 95°C to break the H-bonds between the 2 strands. This exposes the part we want in this lab experiment. Then, the primer binds to the trage we want without cancer marker, this primer won't bind. Next, the temperature will be dropped to 57°C in order to bind the primer. Later, it is heat it back up to 72°C with the Taq to reform and duplicate DNA strands. Finally, this thermal cycling is repeated for amplification and add dye that binds specifically to DNA for detection. | ||
# Pippette 50 μL DNA sample into the labeled PCR tubes. | |||
# Place the pipettes separately on the table to avoid contamination | |||
# Use a pipette to add 50 μL of GoTaq Master mix to each of the PCR tubes, and discard the pipette after each tube. | |||
# Create a new program on the Open PCR system (connected to the computer) | |||
# Create three stages | |||
#* Stage 1: 1 cycle, 95°C for 3 minutes | |||
#* Stage 2: 30 cycles for 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 30 seconds | |||
#* Stage 3. 72°C for 3 minutes | |||
# Final hold: 4°C | |||
# Save the Program | |||
# Place PCR tubes inside the machine and push down on the lid to close it | |||
# Finally, click the start button in the OpenPCR program and run the experiment. | |||
<br> | <br> | ||
Revision as of 17:13, 27 November 2012
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 TEAMLAB 2 WRITE-UPThermal Cycler EngineeringOur re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
Key Features
Instructions
ProtocolsPolymerase Chain Reaction
The Components of the GoTaq® Colorless Master Mix
DNA Samples (8)
DNA Measurement Protocol Research and DevelopmentBackground on Disease Markers
Primer Design
Illustration
|