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 once.
- Cup for discarded tips
- Open PCR machine
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
- Secure all materials required for the PCR
- The strip of 8 empty PCR tubes needs to be cut in half so that two strips of 4 tubes are left.
- Label the sides of the empty tubes with the labels from the table above with a black permanent marker. Use "p" for "+" and "n" for "-" when labeling.
- Put the two strips of 4 tubes in a rack.
- Starting with the positive control, transfer 50 μL of the PCR reaction mix into the empty tube. Make sure to use proper pipetting technique and dispose of the tip once the transfer is completed to ensure there is no cross-contamination.
- Now transfer 50 μL of the positive control/DNA primer mix, using a new pipette tip, into the same positive control tube. The total final volume in the tube should be 100 μL.
- For the negative control follow steps 5 and 6 but use the negative control/DNA primer mix instead.
- For patient 1 replicates 1, 2, and 3 and patient 2 replicates 1, 2, and 3 follow steps 5 and 6 but replace the control DNA primer mix with the correct patient DNA primer mix.
- All eight tubes should have 100 μL of complete PCR reaction in each tube.
- Make sure that all lids are closed tightly.
- Using the assigned PCR machine place the tubes into the slots in the heating block. Two groups should use one PCR machine as 16 filled slots are necessary before running the machine. Once both groups have placed their tubes into the machine it is okay to start.
HEATED LID: 100°C
INITIAL STEP: 95°C FOR 2 minutes
NUMBER OF CYCLES: 35
Denature at 95°C for 30 seconds, Anneal at 57°C for 30 seconds, and Extend at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
Research and Development
PCR - The Underlying Technology
Q1) What is the function of each component of a PCR Reaction?
In a PCR Reaction there are several components necessary for the reaction to take place, the template DNA, Primers, Taq Polymerase, and Deoxyribonucleotides, or dNTP's.
The template DNA is the original strand of DNA which hosts the DNA fragments that are going to be copied by the PCR Reaction.
The primers bind to the specific sites on the template DNA to denote which parts of the DNA are to be copied by the PCR reaction. These primers are sequences of nucleotides that are designed only to bind with the specific DNA sequence within the template DNA. Two primers are necessary for the duplication of DNA, one for each strand of DNA.
The Taq Polymerase, an enzyme derived from a thermophilic bacterium, is the enzyme that duplicates the specific section of the template DNA as denoted by the primers. It does this by binding to the start primer, then moving down the template DNA, adding the nucleotides that compliment the ones on the original strand.
The dNTP's are the monomers that make up DNA. These are necessary for the duplication of the template DNA, as they are what the Taq Polymerase binds to the template strand to duplicate the strand.
Q2) What happens to the components (listed above) during each step of thermal cycling?
INITIAL STEP: 95°C for 3 Minutes:
During the initial heating up of the solution, the Template DNA strands start to denature so that they are two separate strands. In addition, the heating up of the solution activates the Taq Polymerase.
Denature at 95°C for 30 Seconds:
During this step, the DNA template strands finish denaturing completely, so that they do not rejoin during the later steps.
Anneal at 57°C for 30 seconds:
During the annealing step, the DNA Primers are attached to the specific sites on the DNA template strand.
Extend at 72°C for 30 seconds:
During the extending step, the Taq polymerase binds to the DNA primers. After that, they move along the DNA template strand, adding the dNTP's onto the template strand.
FINAL STEP: 72°C for 3 Minutes:
The solution is maintained at extension temperature for a period of time after the extension step to ensure that all strands that were being extended are completely extended.
FINAL HOLD: 4°C:
The solution is then brought to a temperature close to freezing so that activity from the taq polymerase is limited, as the taq polymerase as active at higher temperatures.
Q3) DNA is made up of four types of molecules called nucleotide, designated as A, T, C, and G. Base-pairing, driven by hydrogen bonding, allows base pairs to stick together. Which base anneals to each base listed below?
Adenine -> Thymine
Thymine -> Adenine
Cytosine -> Guanine
Guanine -> Cytosine
Q4) During which two steps of thermal cycling does base-pairing occur? Explain your answers.
Base pairing occurs during the steps in which the primers are attached to the DNA Template strand, as well as during the steps during which the taq polymerase attaches the dNTP's to the DNA template strand. These actions can only take place during these steps because they are held at temperatures at which the DNA strands are denatured, but the temperature is not too high where no hydrogen bonds can occur.