BME100 f2013:W900 Group10 L4

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(Protocols)
(Protocols)
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Step 8: After the machine has finished store the PCR tubes for later use.
Step 8: After the machine has finished store the PCR tubes for later use.
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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.
 
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Step 2:
 
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Pour the reaction mix into the the eight test tubes of the thermal cycler where the DNA fragments are located. Securely close the thermal cycler and start the reaction.
 
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Step 3:
 
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Dispose of the used pippets.
 

Revision as of 03:21, 30 October 2013

BME 100 Fall 2013 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name: Barrett Anderies
Name: Barrett Anderies
Name: Joslin Jose (Protocol)
Name: Joslin Jose
(Protocol)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)
Name: studentRole(s)
Name: student
Role(s)

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.


Experimenting With the Connections

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.


Test Run

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.




Protocols

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 copying.

- Step 7: Bring the temperature down to 4 degrees celsius to stabilize PCR mixture.


DNA Sample Set-up

Image:bme_100group10Lab4.PNG

DNA Sample Set-up Procedure

Step 1: Plug in and turn on OpenPCR machine.

Step 2: Transfer the 8 DNA samples into their respective PCR tube, making sure to use a new pipette each time.

Step 3: Transfer primer mixture into each PCR tube.

Step 4: Transfer PCR reaction mixture into into each of the PCR tubes.

Step 5: Open the OpenPCR and place all 8 PCR tubes into the OpenPCR machine.

Step 6: Program the OpenPCR machine to follow the steps outlined in "Thermal Cycler Program" above.

Step 7: Close the OpenPCR and run the program.

Step 8: After the machine has finished store the PCR tubes for later use.


PCR Reaction Mix

The PCR reaction mixture contains 50µl of each of the following:

- Taq DNA polymerase - responsible for the assembly of the new DNA stands

- MgCl2 - a co-factor for the Taq DNA polymerase. Is a catalyst and is therefore not consumed by the reaction.

- dNTP's - the building blocks of DNA (Adenine, Thymine, Guanine and Cytosine). Required to construct the new DNA strands.


DNA/ primer mix

The DNA/primer mixture contains 50µl of each of the following:

- DNA template - the DNA molecule containing the sequence we are trying to amplify.

- Forward primer - the short single strands of DNA that is complementary to the starting sequence of the segment we are trying to copy.

- Reverse primer - the short single strands of DNA that is complementary to the ending sequence of the segment we are trying to copy.




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.



(Add a write-up, essay-style, organized into paragrpahs with descriptive headers, based on the Q&A's from Section three of your worksheet)

(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.)





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