Difference between revisions of "BME100 f2013:W900 Group10 L4"
(→Research and Development)
|Line 53:||Line 53:|
'''Thermal Cycler Program'''
'''Thermal Cycler Program'''
for DNA. of .
- to .
72 the polymerase. of cycle.
: the down .
Revision as of 22:56, 29 October 2013
|BME 100 Fall 2013|| Home |
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Wiki Editing Help
LAB 1 WRITE-UP
Initial Machine Testing
The Original Design
OpenPCR is a small and relatively low cost PCR (polymerase chain reaction) machine. In the past most PCR machines were prohibitively expensive, and thus not available to most schools and enthusiasts. The designers of OpenPCR changed this by creating a very small machine containing low cost and widely available electronics (Arduinos, theremometer, LCD, ect.) and other components (wooden frame, etc.). A polymerase chain reaction, or PCR, is a chemical reaction that takes advantage of biological molecules (mostly proteins) to make billions of copies of a certain segment of a template DNA strand. A PCR reaction relies on accurately timed temperature variation to work properly. This is what a PCR machine is for. Tubes containing the PCR reactants are placed into the the heat exchange block in the PCR machine. The machine then proceeds to cycle the temperature of the PCR mixture for a user defined period of time and over a user defined temperature range. The result is massive amplification of the desired DNA segment in the PCR reaction tubes.
When we unplugged the LCD screen (part 3) from Arduino and PCR shield (part 6), the LCD screen turned off because it was no longer receiving power or signal from the main circuit board.
When we unplugged the white wire that connects the Arduino and PCR shield (part 6) to the thermometer (part 2), the machine began displaying incorrect temperature information.
On October 23, 2013, we ran the PCR machine through a test cycle. The machine ran smoothly throughout the entire process (10:06am - 11:24am) and ran very closely the the rate estimated by the time to completion readout. At all points during the test the information displayed on the PCR machine's LCD screen matched that displayed on the computer.
Thermal Cycler Program
- Set heating lid temperature to 100 degrees celsius.
- Step 1: Heat to 95 degrees celsius for 3 minutes to fully denature all DNA.
- Step 2: Hold at 95 degrees celsius for 30 seconds to denature DNA. (First step of amplification cycle).
- Step 3: Hold at 57 degrees celsius for 30 seconds to allow annealing (primers attach).
- Step 4: Hold at 72 degrees celsius for 30 seconds for extension (copying of the bracketed area by Taq polymerase). (Last step of amplification cycle).
- Step 5: Repeat steps 2 - 4 thirty five times.
- Step 6: Hold at 72 degrees celsius for 3 minutes to ensure the Taq polymerase has finished
- Step 7: Bring the temperature down to 4 degrees celsius to stabilize PCR mixture.
DNA Sample Set-up Procedure
Step 1: Gather the materials necessary to conduct the lab. First extract the eight samples of the desired DNA fragments and place it into the eight different test tubes in the thermal cycler. Then gather the eight samples of 50 micro-liters of the PCR reaction mix. This mix should contain Tag DNA polymerase, MgCl2, template DNAs, Primers (forward and reverse), and dNTPs.
Research and Development
PCR - The Underlying Technology
In the first cycle, the temperature is raised to 95 degree Celsius which is nearly boiling point. This is when the double helix DNA separates and forms two single strands of DNA. Later on, the thermal cycler cools to about 50 degree Celsius. This causes the two single stand DNAs to join back together. However, there are a lot of primer sequences floating around due to the reaction mix. The primer sequence then grips on to the single stranded DNA and locks to it. This causes the single stranded DNA to not join with the other strand and stay separated. After that, the thermal cycler heats up to 72 degrees Celsius. This activates the tag DNA polymerase’s. Tag DNA polymerase find the end of the primer and starts to attach complimentary DNA nucleoids to both of the single stranded DNA. This is what causes the replication of DNA to take place. This marks the end of cycle two. In cycle three there are two identical DNA strands and as the cycle continues the replication continues. By the end of the cycle 30 there would be about a billion copies of DNA strand.
Is an affordable tool that is used to focus on a specific segment of DNA and produces about a billion copies. It is used in numerous ways such as for medical research, identifying a marker gene, diagnosing a disorder, identifying bacteria, and crime scenes. The thermal cycler program work by gathering desirable DNA and placing it in the PCR tubes. The tubes then are lowered into the temperature chamber using the top part of the PCR machine. Then the cycler raises and lowers temperature according to the pre-set program. The program contains thirty cycles and takes about two hours to complete. In the first cycle, the thermal cycler heats up to 95 degree Celsius which is almost boiling point. During this time, the DNA double helix spates forming two single strand DNA. Then the thermal cycler cools to about 50 degree Celsius. At this time the single stranded DNA would want to match up with its original pair. However, the primer sequences lock on to the single stranded DNA before it can join. The thermal cycler then changes temperature to 72 degree Celsius which activates the DNA polymerase. DNA polymerase then finds the primers and starts attaching complimentary nucleotides to the DNA strand until it reaches the end. This marks the end of cycle one and start of cycle two. In the second cycle, the same steps repeat. In cycle three, the desired product appears: two strands that begin with primer one and end with primer two. Even though it is only two strands during this time, the strands makes more and more copies further down the cycles. By the end of cycle four there will be eight fragments and twenty two by cycle five etc. After thirty cycles there will be over a billion fragments.
(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)