Name: Dominique Stewart
Name: Larrison Black
LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
- DNA/primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
- A strip of empty PCR tubes
- Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
- Cup for discarded tips
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
||PCR Reaction Sample
||Patient 1, replicate 1
||Patient 1, replicate 2
||Patient 1, replicate 3
||Patient 2, replicate 1
||Patient 2, replicate 2
||Patient 2, replicate 3
DNA Sample Set-up Procedure
- Divide the 8 PCR tubes in half
- Label tubes according to DNA/primer mix (positive, negative, patient 1 replicate 1, patient 1 replicate 2, ...)
- Place tubes in the rack
- Change the setting on the micropippettor to 50μL
- Transfer 50μL of PCR reaction mix into the tube which was labeled as the positive control
- Dispose used pipette tip
- Insert new pipette tip
- Transfer 50μL of positive control DNA/primer mix into the tube labeled positive control.
- Close the lids of the tubes tightly
- Set-up the PCR machine according to the instructions located in the "Open PCR program" section.
- Place tubes into the slots in the heating block of the PCR thermal cycler machine
- When all 16 slots are filled (multiple groups use one PCR) run the program
- Wait until the conclusion of the PCR program
- Record all data
- Return reusable materials
- Dispose waste such as used tubes, gloves, pipette tips, etc. into the Biohazard Bag
- Clean table
The following information should be inputted into the PCR machine in order to set it up for the experiment according to the Manual:
- Heated Lid: 100°C
- Initial Step: 95°C for two minutes
- Number of Cycles: 35
- Denature at 95°C for 30 seconds
- Anneal at 57°C for 30 seconds
- Extend at 72°C for 30 seconds
- Final Step: 72°C for two minutes
- Final Hold: 4°C
Polymerase chain reaction is commonly known by itʻs abbreviation: PCR. PCR is a method of exponentially replicating DNA molecules to easily detect and test the DNA for human diagnostics, environmental monitoring, and scientific research. PCR are consisted of four main components: Template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides (dNTPʻs). The template DNA are the initial double stranded DNA sequence before going through the PCR process. In correspondence to the lab conducted in class, the template DNA would be the 16 tubes (8 from one group and 8 from another). Primers are another component of the PCR reaction which can be defined as short, custom pieces of DNA to make enzymes known as primase. Two primers are required to replicate DNA because the double stranded DNA separates at a high temperature and becomes two single stranded DNA. Each primer attaches to each strand of DNA in order to replicate the DNA. DNA Polymerase is another component of the PCR reaction that has two main functions. The first function is replicating a cellʻs DNA prior to the cell dividing into two and the second function is attaching a primer to a base-pair with a longer piece of DNA. Base-pairs are particular pairs of nucleotides known as Deoxyribonucleotides (dNTPʻs). The dNTPʻs are Adenine (A) and Thymine (T) paired together and Cytosine (C) and Guanine (G) paired together. Each single nucleotide are known as “building blocks of DNA,” which is what the Primers and DNA Polymerase are replicating and building. Base-pairs are the groupings of the four nucleotides so A will always be with paired with T and likewise for C and G as a result of hydrogen bonding. The more commonly used DNA Polymerase when dealing with PCR is called Taq Polymerase due to the high temperature it can withstand (72ºC or 161ºF) making this DNA practically heat resistant. As previously mentioned with DNA Polymerase, Taq Polymerase generates new strands of DNA by replication and are short pieces of single stranded DNA.
In a polymerase chain reaction, the machine conducts most of the work regarding the thermal cycle. Once the template DNA is put into the machine and the program settings are inputted, the first step of the thermal cycle is heating the template DNA to 95ºC for three minutes. The next step is called Denature or Denaturation, which is the process when the DNA separates into two complementary single strands from the one double stranded DNA. This phase lasts three minutes and then switches to the Anneal or Annealing phase, which has a lower temperature of 57ºC and also lasts thirty seconds. During this phase, the two primers bind to the complementary single strands. Next, the phase called Extend occurs at 72ºC and that lasts for thirty seconds where the Taq Polymerase generates new strands of DNA and attaches the primer to the base-pair. The next step continues to make the DNA larger with the replication of the DNA by the Taq Polymerase for three minutes. These steps are repeated 35 times, hence the name “thermal cycle.” Lastly, when the cycles are complete, the machine lowers the temperature to 4ºC to maintain the “product integrity.” This last step is called the Final Hold and stays that way till the tubes are removed.
There are four types of DNA nucleotides adenine, thymine, guanine, and cytosine. These four nucleotides are necessary for base-pairing during the polymerase chain reaction and are paired through hydrogen bonding. After the denaturation of the double-stranded DNA tha pairing begins and adenine bonds to thymine and cytosine bonds to guanine during the annealing phase. The same base pairing occurs during the extension part of of the polymerase chain reaction.
The image above shows the PCR process and also displays the exponential amplification of DNA.
First, in a polymerase chain reaction, the double stranded DNA is heated to approximately 95 degrees Celsius causing the DNA to denature into two separate strands. Then, the reaction is slightly cooled allowing primers to anneal to each strand and copy the targeted DNA sequence. Finally, an enzyme(Taq Polymerase) attaches itself to the site of the primers targeted sequence and adds nucleotides to create the second strand. These steps are repeated to create more copies of the DNA sequences.