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 and reverse primer.
- Strip of empty PCR tubes
- Disposable pipette tubes
- Cup for discarded tips
- OpenPCR 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
- Gather all materials listed in the protocol section of Lab A.
- Assemble two strips of four linked PCR tubes, by cutting the PCR tubes in half.
- Label the sides of the PCR tubes with a black maker.
- Align the PCR tubes on a rack.
- Using a micropipettor, transfer 50uL of the PCR reaction mixture into the tube labeled "positive control".
- Discard the disposable tip of the micropipettor, replacing it with a new one to prevent cross contamination.
- Add 50uL of positive control DNA/ primer mixture into the positive control PCR reaction tube.
- Repeat steps 5-7 for the negative control, patient 1 replicates 1, 2, and 3, and patient 2 replicates 1, 2, and 3. All the tubes should have 100uL of solution by the end of this step.
- Ensure that the lids to the PCR tubes are tightly closed.
- Place all eight PCR tubes into the designated PCR machine. Be mindful to wait for another group to completely fill the sixteen slots available in the PCR machine. Once all slots are filled ask a TA for the okay to start the run.
HEATING LID: 100 °C
INITIAL STEP: 95 °C for 2 minutes
NUMBER OF CYCLES: 35
Denatures at 95 °C for 30 seconds, Anneal at 57 °C for 30 seconds, and Extend at 37 °C for 30 seconds
FINAL STEP: 72 °C for 2 minutes
FINAL HOLD: 4 °C
Research and Development
PCR - The Underlying Technology
The PCR Reaction
The four components necessary in a PCR reaction are the template DNA, the primers, taq polymerase, and dNTP's (deoxyribonucleotides). The template DNA is the DNA from the subject that will be duplicated in the PCR reaction. The primers are short pieces of DNA that are made in the lab which will attach to the segments of DNA that are to be copied. The taq polymerase is a naturally occurring protein that attaches to the primers and begins adding nucleotides to the DNA and the dNTP's are A's, C's, G's, and T's that are needed to build the DNA.
The Steps of Thermal Cycling
Thermal cycling has 6 steps: Initial, Denature, Anneal, Extend, Final, and Final Hold. Three of theses steps (denature, anneal, and extend) are repeated anywhere between 20 and 35 times allowing for the desired about of DNA replication. During the initial step the temperature is held at 95°C for 2 minutes activating the heat activated polymerases. Next during the denature step the solution is heated to 95°C for 30 seconds to denature the DNA. Denaturing causes the bonds between the complementary paired bases of the two DNA strands to be disrupted disrupted allowing the two DNA strands to separate. The denature step is followed by the anneal step during which the solution is cooled from 95°C to 57°C for 30 seconds allowing the primers to anneal or attach to the DNA this in turn allows the polymerase to attach to the primers. For the extend step the temperature is once again raised to 72°C which is the optimal activity temperature for the Taq polymerase enzyme. During this step the Taq polymerase synthesizes a copy of the DNA strand by adding complementary bases. At this point in the process the previous 3 steps are repeated in a cycle till the desired amount of DNA is present. Following this cycle the final step allows any remaining single stranded DNA to be fully extended by holding the temperature at 72°C for 3 minutes. Finally During the final hold step the temperature is lowered to 4°C for an indefinite amount of time to allow for short-term storage of the reaction.
Base pairing occurs during the annealing and the extending steps in Thermal Cycling. During Base pairing, a primer attaches itself on the top strand, at one end, of the DNA segment of interest, and another primer attaches to the bottom strand, at one end, of the DNA segment. This is possible due to the separation of the DNA helixes during the denaturing step. The primers “anneal” or attach themselves to the appropriate binding site on the DNA strands. Because the DNA bases always pair with their complementary bases [A; T and G; C], the primers link to the specific desired binding sites on the DNA strands. After the temperature is raised to 72 degrees, the extending step occurs and the DNA polymerase seals the primers in place in the 5' to 3' direction of the strands.