PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl 2, 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 only once. Never reuse disposable pipette tips. If you do, the samples will become cross-contaminated
Cup for discarded tips
Micropipettor
OpenPCR machine: shared by two groups
PCR Reaction Sample List
Tube Label
PCR Reaction Sample
Patient ID
G5 +
Positive control
none
G5 -
Negative control
none
G5 1-1
Patient 1, replicate 1
G5 1-2
Patient 1, replicate 2
G5 1-3
Patient 1, replicate 3
G5 2-1
Patient 2, replicate 1
G5 2-2
Patient 2, replicate 2
G5 2-3
Patient 2, replicate 3
DNA Sample Set-up Procedure
Label each of the 8 tubes with the names listed above
Pipette 50 µL of the PCR reaction mix into the positive control tube
Dispose of the pipette tip to avoid cross-contamination
Pipette 50 µL of the DNA/primer mix into the positive control tube (total volume in tube is now 100 µL)
Repeat steps 2-4 for the rest of the tubes with designated DNA/primer mix for each
Close the lids on tubes and insert them into thermal cycler
OpenPCR program
Heated Lid: 100 °C.
Initial Step: 95 °C for two minutes.
Number of Cycles: 25, 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 two minutes.
Final Hold: 4 °C.
Research and Development
PCR - The Underlying Technology
Components on a PCR Reaction
Template DNA: One strand of DNA used as a template to create a complementary strand. Because of this, one double stranded DNA molecule is converted into two, and both strands are identical to the first. When the DNA molecule is denatured in two single strands, each of this become the template for the PCR process.
Primers: Primers are short DNA or RNA sequence that mark the beginning point for DNA synthesis. Specifically, the bind to the 5-end of the template strand and Taq Polymerase binds to them to begin the DNA synthesis.
Taq Polymerase: The enzyme (protein) that assembles nucleotides into new strands of DNA.
Deoxyribonucleotides (dNTP’s): Single units of bases A, T, G, C
Components during each step of the thermal cycling
Initial Step: 95°C for 3 minutes:
Template DNA: Separates into two strands. Primers: The primers stay in the solution. They don’t interfere in the DNA molecules yet. Taq Polymerase: Not activated at this stage dNTP’s: The nucleotides bases stayed in the solution. They don’t interfere in the DNA molecules yet.
Anneal at 57C for 30 seconds:
Template DNA: The two single strands tend to go back together but the abundance of primers in the tube prevents them of doing this. Primers: Primers bind to the respective. complementary sequences in both single-strands at their 5-end. Taq Polymerase: Taq Pol is not activated at 57°C. dNTP’s: The nucleotides bases stayed in the solution. They don’t interfere in the DNA molecules yet.
Extend at 72°C for 30 seconds:
Template DNA: DNA is now bind by the primers and Taq Polymerase, ready to be synthesized. Primers: One Taq Polymerase protein binds to each primer. Taq Polymerase: One complex binds to the end of the primer. dNTP’s: Taq Polymerase begin to add them to the single-strands that are going to be synthesized.
Final Step: 72°C for 3 minutes:
Template DNA: Each single-strand is synthesized in a copy of the original double stranded DNA molecule. Primers: N/A Taq Polymerase: Adds the nucleotides creating the second complementary strand. dNTP’s: Nucleotides are added by Taq Polymerase matching respectively their complementary bases from the template single-strand.
Final Hold: 4°C:
Template DNA: There is now two DNA molecules for each original template DNA at each PCR cycle, and the synthesized molecules are stabilized at the low temperature. Primers: Remain in the solution if they were not the limiting reactant for the PCR. Taq Polymerase: Remain in the solution if they were not the limiting reactant for the PCR. process. dNTP’s: Remain in the solution if they were not the limiting reactant for the PCR.
Nucleotide Base Pairing
Adenine (A): Thymine (T).
Thymine (T): Adenine (A).
Cytosine (C): Guanine (G).
Guanine (G): Cytosine (C).
Base Pair Occurrence
Stage Occurrence: Base-pairing occurs at the annealing step when the primers bind to the DNA strand. It also occurs at the extension step, when the DNA polymerase adds complementary base pairs to the target strand.
BME 100 Spring 2017
