DNA/primer mix, 8 tubes, 50uL each: Each mix contains a different template DNA. All tubes have the same forward primer and the reverse primer
A strip of empty PCR tubes
Disposable pipette tips: only use each once. Never re-use disposable pipette tips or samples will be cross-contaminated
Cup for discarded tips
Micropipettor
Open PCR machine:shared by two groups
Thermal Cycler Program
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G33 +
Positive control
none
G33 -
Negative control
none
G33 1-1
Patient 1, replicate 1
31366
G33 1-2
Patient 1, replicate 2
31366
G33 1-3
Patient 1, replicate 3
31366
G33 2-1
Patient 2, replicate 1
26606
G33 2-2
Patient 2, replicate 2
26606
G33 2-3
Patient 2, replicate 3
26606
DNA Sample Set-up Procedure
1. Set the micro pipette to 50 micro-liter
2. Attach a fresh micro pipette tube to the micro pipette pump.
3. Draw Patient DNA of 50 micro-liters
4. Deposit Patient DNA into empty PCR container
5. Dispose of micro pipette tip
6. Attach a fresh micro pipette tube to the micro pipette pump.
7. Draw PCR of 50 micro-liters
8. Deposit PCR of 50 micro-liters into empty PCR container
9. Dispose of micro pipette tip
10. Placing the tubes in the Thermal Cycler
11. Repeat steps 1-10
PCR Reaction Mix
Component 1
Component 2
Component 3...
DNA/ primer Mix
Component 1
Component 2
Component 3...
OpenPCR program
The following values will be used to run a heating and cooling program on the thermal cycler.
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
Section Two: Research and Development
PCR - The Underlying Technology
Functions of a PCR Reaction
Template DNA: a sample of DNA that contains the targeted sequence
Primers: short pieces of single-stranded DNA that are complementary to a targeted sequence, allows polymerase to begin synthesizing new DNA
Taq Polymerase: a heat stable enzyme used in PCR to amplify DNA segments
Deoxyribonucleotides (dNTP’s): single units of nucleotides (A, G, T, and C), building blocks for new DNA strands
The Process of Thermal Cycling
Initial Step: 95 ℃ for 3 minutes, heated to near boiling
Denature: 95 ℃ for 30 seconds, the DNA double helix separates to create two separate strands of complementary DNA
Anneal: 57 ℃ for 30 seconds, primer sequences bond to DNA to keep them from rejoining
Extend: 72 ℃ for 30 seconds, the DNA polymerase enzyme is activate and complementary nucleotides are added to the separated DNA strands
Final Step: 72 ℃ for 3 minutes, bonding is secured
Final Hold: 4 ℃, DNA is kept at a cool temperature
Base-Pairing
DNA is made up of four different nucleotides: adenine, guanine, thymine, and cytosine. These four components are the building blocks of DNA and RNA. Adenine is paired with thymine by double hydrogen bonds, and guanine and cytosine are paired by a triple hydrogen bond.
Base-pairing occurs during annealing and extending in thermal cycling.
During annealing, the newly denatured DNA wants to go back to it's original shape of a double helix.Only the most stable strands can do this since they are the desired ones and then they get paired off there. During extending, the DNA polymerase connects at the primers and bonds in the 5' to 3' direction, which is by definition the bonding of the base pairs.