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. Disposable pipette tips should never be reused or samples will be cross-contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G15 +
Positive control
none
G15 -
Negative control
none
G15 1-1
Patient 1, replicate 1
50018
G15 1-2
Patient 1, replicate 2
50018
G15 1-3
Patient 1, replicate 3
50018
G15 2-1
Patient 2, replicate 1
69797
G15 2-2
Patient 2, replicate 2
69797
G15 2-3
Patient 2, replicate 3
69797
DNA Sample Set-up Procedure
Materials mentioned above are obtained from the professors
Two lengths of four tubes should be created by cutting the provided linked tubes in half. Tubes should be empty and will be used for the PCR reaction.
The sides of the tubes should be labeled with a permanent marker then placed in a rack: G15 P (positive control), G15 N (negative control), G15 1-1, G15 1-2, G15 1-3, G15 2-1, G15 2-2, G15 2-3
For the positive control (G15 P), 50 μL of PCR should be pipetted into the empty tube, along with the positive control DNA and DNA primers (50 μL). The total volume within the tube is now at 100 μL. New pipette tips should be utilized after every single transfer of liquids to avoid cross-contamination.
For the negative control (G15 N), 50 μL of PCR should be pipetted into the empty tube, along with the negative control DNA and DNA primers (50 μL). The total volume within the tube is now at 100 μL. New pipette tips should be utilized after every single transfer of liquids to avoid cross-contamination.
For patient one, replicates 1-3 (G15 1-1; G15 1-2; G15 1;3), 50 μL of PCR should be pipetted into each of the empty tubes, along with the patient's DNA and DNA primers (50 μL), Patient ID number 50018. The total volume within the tube is now at 100 μL. New pipette tips should be utilized after every single transfer of liquids to avoid cross-contamination.
For patient two, replicates 1-3 (G15 2-1; G15 2-2; G15 2-3), 50 μL of PCR should be pipetted into each of the empty tubes, along with the patient's DNA and DNA primers (50 μL), Patient ID number 69797. The total volume within the tube is now at 100 μL. New pipette tips should be utilized after every single transfer of liquids to avoid cross-contamination.
Lids should be closed tightly for all tubes, then the tubes should be placed within the heating block of the PCR machine. The machine may be run once sixteen of the slots are utilized.
OpenPCR program
Question 1:
Template DNA: The strand of DNA that is used as a model for replication or the DNA waiting to be copied during the PCR experiment.
Primers: Sequences of nucleotides that bind to specific locations on the template DNA. Primers come in pairs so they can bind on each strand of the DNA.
Taq Polymerase: The enzymes that are actually being replicated based upon the primers that were combined on the template DNA. They bind the first primers and then add necessary nucleotides that complete the original strand as it moves down the template DNA
Deoxyribonucleotides (dNTP's): The monomers that are needed to complete a strand of DNA. They are responsible for the replication of the template DNA and are what the taq polymerase connect with form the strand.
Question 2 and 3:
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
Extend at 72°C for 30 seconds
FINAL STEP: 72°C for 2 minutes
FINAL HOLD: 4°C
First the sample from the template DNA is taken. The template DNA is the original DNA segment that is to be copied. The dNTP’s are the nucleotides of the template DNA. During step one, the PCR tube is kept at 95 degrees celsius and the double helix separates and creates two separate strands. Then the PCR tube is cooled down to 57 degrees and the primers attach to their target sites. “Primers attach to sites on the DNA strand that are on either end of the segment that you want to copy.” The temperature is then increases to 72 degrees and the DNA polymerase is then activated and finds the necessary nucleotide and attaches it to the corresponding strand. Taq polymerase reads the code of a strand and attaches a corresponding nucleotide to create a copy of the DNA. Adeine binds to Thymine, and Cytosine binds to Guanine. The tube is then kept at 72 degrees until the target strand is fully replicated.
Question 4:
Base pairing happens during the annealing period and the extension period. As the system cools off, the target DNA are blinded with the primers so that the replication process can occur. This is part of the annealing process. During the extension stage of the thermal cycle, when the temperature is elevated, is when the DNA comes into existence. In this stage two taq polymerase match with base primers created in the previous stage, which in turn create a new nucleotide stand that sequences DNA. These are the two steps that make DNA replication possible in a PCR model experiment.
BME 100 Spring 2015
