End-Point PCR: Antibody-Mediated Hot Start PCR Protocol with Enhanced Specificity and Yield

Introduction

Why use a hot-start Taq?

As PCR reactions sit at room temperature, during assay setup, nonspecific amplification can occur via:

• Binding of primers to non-specific templates
• Formation of primer dimers, allowing primers to use other primers as templates.

In hot-start PCR, Taq polymerase is inactive until heated. Hot-start PCR activation approaches allow users to minimize non-specific amplification while increasing target yield and specificity.

How does our hot start technology work?

Our JumpStart Taq DNA Polymerase is an antibody inactivated hot-start enzyme. During the initial denature PCR step, Taq DNA Polymerase activity is restored. The resulting PCR exhibits a higher specificity and yield. Unlike chemically modified hot-starts that can take up to 10 min for enzyme activation, antibody mediated hot-start enzymes are activated within 1 min.

Mechanism of antibody mediated hot start PCR

Equipment

• Pipettes dispensing volumes from <1 to 200 μL
• Benchtop microcentrifuge
• Thermal cycler
• Electrophoresis equipment
• UV transilluminator
  

Supplies

• Sterile filter pipette tips
• Sterile 1.5 mL screw-top microcentrifuge tubes (such as CLS430909)
• PCR tubes, select one of the following to match desired format:
  • Individual thin-walled 200 µL PCR tubes (Z374873 or P3114)
  • Strip tubes, 200uL (Z374962)
  • Plates
    • 96 well plates (Z374903)
    • 384 well plates (Z374911)
    • Plate seals
      • AlumaSeal^® 96 film (Z721549)
• dNTP mix, 10 mM each of dATP, dCTP, dGTP, and dTTP (D7295, needed for standard format reagents only)
• Enzyme and buffer, review the following table to define optimal reagents for your application:

• DNA marker, select appropriate marker based upon your PCR amplicon size

• PCR grade water (W1754)
• DNA/cDNA template
  • cDNA reaction diluted 1:10 to detect medium to highly expressed targets or 1:2 to 1:5 for rare transcripts or 10 ng to 100 ng gDNA
• Primers diluted to working concentration (10µM working stocks are sufficient for most assays)
  • Order Custom Oligos here
  • Predesigned gene expression primers are also available for most model organisms (KiCqStart^® SYBR^® Green Primers, KSPQ12012)

Method

1. Leave the DNA polymerase on ice or at -20 ^ºC, thaw the remaining reaction components at room temperature or on ice, vortex to mix, centrifuge briefly and replace on ice.
   
2. Setup PCR reactions
   
   a. Prepare a master mix containing all reaction components with the exception of the DNA/cDNA
   template.
   
   i. Calculate the master mix required by multiply amounts by the number of reactions needed,
   including controls, and then add 10% to ensure a sufficient quantity for all samples.

Note: that Magnesium Chloride is added separately if not already in the PCR buffer or when previous optimisation has revealed a requirement for a concentration.

b. Combine reaction components into a 1.5 mL microcentrifuge tube on ice

3. Mix the master mix by carefully pipetting up and down ensuring that all mix is expelled from the pipette tip, and then pulse or centrifuge briefly to collect the sample at the bottom of the tube.


4. Aliquot 20 μL of master mix into the required number of 200 μL thin-walled PCR tubes (number the samples, including replicates and controls)


5. Add 5 μL of the DNA template sample (containing a total of 10 ng to 100 ng gDNA or dilute a cDNA sample 1:2 to 1:10) to reach a final reaction volume of 25 μL.


6. Spin the PCR tubes and place into a thermal cycler with a heated lid.


7. Determine the appropriate annealing temperature (Ta) for the primers. A good first test can be performed using a Ta that is 5 ^ºC lower than the Tm of the primer with the lowest Tm.


8. Perform the following thermal cycling protocol.
   a. 95 °C for 2-10 min
   b. [95 °C for 30 sec; 48-60 °C (Ta) for 30 sec; 72 °C for 0.5-2 min] 25-50 cycles
   c. 72 °C for 10 min
   
9. Analyse an aliquot of the completed reaction by agarose gel electrophoresis, with visualization on a transilluminator or other chosen analysis method.