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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 Sigma’s hot start technology work?

Sigma's 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:
  • 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:
     
Hot Start DNA Polymerase
Standard format - separate components Readymix- premixed PCR mastermix
Containing MgCl2 Separate MgCl2
(for MgCl2 optimization)
Containing MgCl2
With red dye for direct load on gels Clear formulation
without dye
Clear formulation
without dye
Clear formulation
without dye
With red dye for direct load on gels
JumpStart™ REDTaq® DNA Polymerase
(D8187)
JumpStart™ Taq DNA Polymerase, with MgCl2 
(D9307)
JumpStart™ Taq DNA Polymerase, without MgCl2 (D4184)

JumpStart™ Taq ReadyMix™
(P2893)  

JumpStart™ REDTaq®
ReadyMix™  
(P1107)

 

  • DNA marker, select appropriate marker based upon your PCR amplicon size
     
Product Name (Product Number) DirectLoad™ step Ladder, 50 bp (D3812)
DirectLoad™ PCR 100 bp Low Ladder (D3687)
DirectLoad™ 1kb DNA Ladder (D3937)
Size Range 50bp – 3,000bp 100bp – 1,000bp 500bp -10,000bp
Picture of Ladder
  • 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)

Method

  1. Leave the DNA polymerase on ice or at -20ºC, 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.  

    For Standard Format Hot-Start DNA Polymerases
    Component
    Final Concentration
    (in a 25 µL reaction
    )
    Master Mix Volume
    (µL) for One Reaction

    PCR buffer (10X) 1X 2.5 μL
    Magnesium chloride (50 mM) 2.5 mM 1.25 μL
    Forward/reverse primer (10 μM stock) 50-500 nM Variable
    dNTP mixture (10 mM of each nucleotide) 0.2 mM 0.5 μL
    Taq DNA polymerase (5 U/μL) 0.05 units/μL 0.25 μL
    PCR grade water   
    Up to 20 μL Up to 20 μL
    Total volume (μL)   20 μL


    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.

     

    For ReadyMix Format Hot-Start DNA Polymerases
    Component
    Final Concentration
    (in a 25 µL reaction
    )
    Master Mix Volume
    (µL) for One Reaction

    JumpStart Taq ReadyMix (2X) 1X 12.5 μL
    Forward/reverse primer (10 μM stock) 50-500 nM Variable
    PCR grade water Up to 20 μL Up to 20 μL
    Total volume (μL)   20 μL

     

        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 5ºC 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.

Materials

     
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