LAB 4 WRITE-UP
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 µL each
- DNA/ Primer mix, 8 tubes, 50 µL each
- A strip of empty PCR tubes
- Disposable pipette tips
- A cup for discarded tips
- A micropipetter
- OpenPCR machine
PCR Reaction Sample List
| Tube Label
|| PCR Reaction Sample
|| Patient ID
| G# +
|| Positive control
| G# -
|| Negative control
| G# 1-1
|| Patient 1, replicate 1
| G# 1-2
|| Patient 1, replicate 2
| G# 1-3
|| Patient 1, replicate 3
| G# 2-1
|| Patient 2, replicate 1
| G# 2-2
|| Patient 2, replicate 2
| G# 2-3
|| Patient 2, replicate 3
DNA Sample Set-up Procedure
- Record a list of materials, samples and the tube labels.
- Cut the strip of empty PCR tubes in half so that you have two strips of four linked tubes. This is necessary to fit all of your tubes into the OpenPCR machine.
- Use a black marker to label the sides of the empty tubea.
- Place the tubes in a rack.
- Starting with the empty tube labeled as the positive control, transfer 50 μL of PCR reaction mix into an empty tube.
- Transfer the positive control DNA/ primer mix into the same tube.
- Repeat steps for the negative control, patient 1 and patient 2 samples.
- Close the lids on PCR reaction tubes.
- Place tubes into PCR machine.
Research and Development
PCR - The Underlying Technology
What is the function of each component of a PCR reaction
- Template DNA: A desired segment of DNA intended for amplification.
- Primers: Laboratory made DNA segments designed to bind to specific DNA sequences for amplification.
- Taq Polymerase: An enzyme that anneals to primers and begins to create a new complementary strand of DNA by adding nucleotides.
- Deoxyribonucleotides: Building blocks of DNA used by Taq Plymerase to complete the complementary DNA strand.
What happens to the componenets (listed above) during each step of thermal cycling?
- INITIAL STEP - 95°C for 3 minutes: As the temperature is raised to 95°C, the enzymes in the PCR reaction are activated for DNA amplification and to unwind all complex double helix DNA structures in a sample. http://www.sigmaaldrich.com/technical-documents/articles/biology/polymerase-chain-reaction.html
- Denature at 95°C for 30 seconds: The DNA strands separate(denature) into single strands called template DNA.
- Anneal at 57°C for 30 seconds: Primers bind(anneal) to predesignated regions on the template DNA, indicating areas intended for amplification.
- Extend at 72°C for 30 seconds: Taq polymerase attach to the primers on the template DNA and proceed to create a complementary strand of DNA using nucleotides.
- FINAL STEP-72°C for 3 mintues: The final three minutes are used to ensure every possible strand of DNA has been extended during the amplification process.
- FINAL HOLD - 4°C: The temperature of 4°C is used to deactivate the taq polymerase enzyme. http://www.nyxtechnik.com/en/support.asp?type=faq
DNA is made up of four types of molecules called nucleotides, 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(A) : Thymine(T)
Thymine(T) : Adenine(A)
Guanine(G) : Cytosine(C)
During which two steps of thermal cycling does base-pairing occur? Explain your answers.
Base-pairing occurs during the extended and final step of the PCR reaction. AT 72°C, the enzyme taq polymerase is at it's optimal temperature and proceeds to attach to primers on template DNA strands. Once attached, the enzyme attains the proper base pairs to create a complementary DNA strand for amplification.