BME100 f2013:W1200 Group18 L4

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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: Carlyn HarrisRole(s):Protocol
Name: Carlyn Harris
Role(s):Protocol
Name: Tori PlattRole(s):Reseach and Development
Name: Tori Platt
Role(s):Reseach and Development
Name: Nikhil PatelRole(s):Initial PCR Testing
Name: Nikhil Patel
Role(s):Initial PCR Testing
Name: Alexandra OlsonRole(s): Protocol
Name: Alexandra Olson
Role(s): Protocol
Name: Matthew ArmasRole(s):Initial PCR Testing
Name: Matthew Armas
Role(s):Initial PCR Testing

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
Image:PCR Machine.png

Tis is a PCR machine. The sample holder is at the top of the machine, this is where we will place tubes with samples in them to be held in place by holes in the square. The heating lid covers the sample holder, it is there to keep the heat around the sample holder. The heater inside the PCR machine will heat up the sample holder. The fan is there to keep the inside of the PCR machine cool, because there are electronics that can be damaged from over heating. The circuit board keeps the sensors active in a screen on top of the PCR machine, it tellls us how hot the heater will be, how long the machine will run for the experiment, and other data as well.


Experimenting With the Connections

When we unplugged (part 3) from (part 6), the machine's screen on top of the box no longer functioned. The screen shows the data of the experiment, but because it was unplugged, we could not see the data.

When we unplugged the white wire that connects (part 6) to (part 2), the machine's heat sensor no longer functioned. It determines how hot the machine runs, therefore the machine did not pick up any heat when it was unplugged.

Test Run

Open PCR was first tested on 10/23/13 from 1:18pm to 1:44pm. During the lab, we inspected the 16-tube PCR block and the heating lid, which are both designed to retain a constant temperature across the samples that would be tested. We also explored the insides of the machine, and how its various components affected one another. Our experience with the machine was somewhat frustrating because the machine failed to complete the required amount of cycles in a reasonable amount of time. It was labeled as being slow.




Protocols

Thermal Cycler Program
Lid heated to 100 degrees Celsius
1.Initial: Thermo-cycler reaches 95°C for 3 minutes. Here, the DNA double helix separates into 2 strands
2. 35 cycles of the following:
-Denature at 95°C for 30 seconds. Double helix separates.
-Anneal at 57°C for 30 seconds. Primers attach to their target strands
-Extend at 72°C for 30 seconds.DNA polymerase is activated. It locates the primer and begins to add complementary nucleotides onto the strand until it gets to the end of the strand.
At the end of these steps, you have numerous replicates of your target DNA
3. Final Step: 72°C for 3 minutes
4. Final hold: 4°C


DNA Sample Set-up
PCC= Positive control: Cancer DNA
NCC= Negative control: Non-cancer DNA
P11,P12,P13= Patient 1's DNA samples
P21,P22,P23= Patient 2's DNA samples
Patient 1 ID: 54174
Patient 2 ID: 43184


PCC P11 P12 P13
NCC P21 P22 P23


DNA Sample Set-up Procedure
1. Label 6 50 μL disposable pipette tips with the labels given below corresponding to each patient.
2. Label the remaining 2 50 μL pipette tips, one as the positive control cancer DNA (PCC) and one as the negative control non-cancer DNA (NCC).
3. Pipette the PCR reaction mix into each reaction tube.
4. Using a different disposable pipette tip for each sample, pipette the DNA sample primer mix into each reaction tube.
5. Place the reaction tubes in the thermal cycler.
6. Run the PCR software that follows the protocol below.


PCR Reaction Mix

The PCR reaction mix contains:

   -Tag DNA Polymerase
   -MgCl2
   -dNTP's


DNA/ primer mix

The DNA primer mix contains:

   -Different template DNAs
   -Forward primer
   -Reverse primer





Research and Development

Polymerase Chain Reaction (PCR) - The Underlying Technology

Polymerase chain reaction occurs when DNA is heated and cooled at exact temperatures for exact intervals of time. However, there are five components required for the reaction to work successfully; template DNA, primers, TAQ polymerase, Magnesium Chloride (MgCl2), and dNTPs (deoxyribonucletides).

In order to replicate a section of DNA, a source for the sequence needs to be present for the primers to attach to. The template DNA provides the original copy to begin the entire process. What PCR enables is the replication of a target sequence in DNA that is made thousands of times over.

Once a target DNA sequence is established, primers are responsible for marking the beginning and end of these sequences in order for them to be coded in the right direction (5'-3') by Taq Polymerase. Primers are short strands of oglionucleotides that tell the enzyme where to stop and start.

After primers have attached themselves to the template DNA, an enzyme called Taq Polymerase binds to the initial 5' primer and moves down the 3' direction to the 3' primer. The role of Taq Polymerase in the PCR process is to code the new strands of DNA so that the new strands compliment the previous strands with the complimentary bases that match with the original template. However, the enzyme will not function without the presence of Magnesium Chloride.

Magnesium chloride is found in the PCR as a catalyst for the Taq Polymerase enzyme. It's not consumed by any aspect of the reaction, but necessary. An abundance of magnesium chloride does speed up the process of the reaction, yet can cause strands of DNA was not what was supposed to be the desired copied sequence. Without a substantial amount of Magnesium chloride, the PCR cannot continue to replicate. Therefore, not as many desired copies will be obtained at the end of PCR.

Deoxyribonucleotides (dNTPs) are essentially the building blocks of DNA. In the solution, unpaired bases float around waiting to be used up. Each nucleotide base pairs with one and only one other base. For example, guanine only pairs with cytosine while thymine only pairs with adenine in double stranded DNA. After Taq Polymerase latches itself onto the location of the primers on template DNA or a previously created strand, it beings to attach dNTPs to compliment whichever base pair comes up as it goes along the sequence. In the Polymerase Chain Reactions, deoxyribonucleotides are present for new strands of DNA to be created throughout the process.

The first step of PCR is the heating up of DNA to 95°C for three minutes; once at 95°C, thirty seconds is allowed for the DNA to begin the PCR process. When doing so, the DNA denatures, separating the two strands that make up the double-helix structure. Hydrogen bonds between nucleotides that keep the helix bound together essentially melt away as the DNA becomes extremely heated. In the next step at 57°C for 30 seconds, primers anneal themselves to the target strands on the template DNA in the appropriate forward and reverse regions. As the proccess goes along, reverse and forward primers attach to previously made DNA sequences along with the template DNA. TAQ Polymerase grabs a hold of the primer areas to begin filling in the complimentary sequence once the mixture is heated back up to 72°C in the next phase. Magnesium chloride acts as a catalyst for this aspect of the reaction. Once TAQ Polymerase reaches the ending primer, it detaches itself and the reaction starts all over again at the denaturing of DNA strands. This occurs about 35 times over the course of just over an hour.

In the DNA, cancer has its own code. By amplifying certain sequences of the DNA, a common cancerous sequence can be sought out by the primers considering that they are designed to bind to only cancerous stings of nucleotide mutations.The image below shows the binding of primers to complimentary combination of the oglionucleotide. With isolating the cancerous strand, it makes it easier to work with and study rather than having to look at the entire genome that was originally placed in the sample.
Image:EssGen4-4 PrimersAnneal MID.jpg

To elongate the strand of target DNA, TAQ Polymerase matches up base pairs as shown below. dNTPs come to fill in the matching pair (G to C, T to A) and the strand is elongated into a mutated cancerous strand of DNA. Over the time period in which PCR occurs, several hundred strands of cancerous DNA have been created exponentially.

Image:Pcrstep3.gif





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