Introduction

Description

The PCR Reagent System provides qualified reagents for the amplification of nucleic acid templates by Polymerase Chain Reaction (PCR). The system contains sufficient reagents suitable for 100 amplification reactions of 100 μL each. PCR is a method for exponential amplification of nucleic acids in vitro by repeated thermal cycling (1,2). In a typical cycle, the double-stranded DNA is first heat-denatured at a high temperature (~95°C), then annealed at a lower temperature (~50°C) to two oligonucleotide primers that are complementary to a specific region of the template and finally extended at an intermediate temperature (~72°C) by Taq DNA Polymerase. This cycling is repeated several times resulting in ~105-109 fold amplification of the defined segment of target DNA (1,3)


Section 1.01
Component Part No.
Amount
10X PCR Buffer plus Mg: [200 mM Tris-HCl (pH 8.4), 500 mM KCl, 15 mM MgCl2] Y02255
1.2 mL
10X PCR Buffer minus Mg: [200 mM Tris-HCl (pH 8.4), 500 mM KCl]
Y02260
1.2 mL
50 mM MgCl2
 Y02277
500 μL
10 mM dNTP Mix: [10 mM each dATP, dCTP, dGTP, dTTP]
Y02256 250 μL
Taq DNA Polymerase (5 U/μL)
Y02257
50 μL
Control DNA (human genomic DNA isolated from K562 cells; 200 ng/μL)
Y02258
3 μg
Control Primer Mix (10 μM each)
Y02259
50 μL


Taq DNA Polymerase Storage Buffer

20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 50% (v/v) glycerol, Stabilizers

Control DNA

High molecular weight human genomic DNA isolated from K562 cells serves as the system control. This DNA can also be used to test any set of gene-specific primers.

Control Primer Mix

The primer mix contains a mixture of sense and anti-sense primers, at a concentration of 10 μM each. These primers are specifically designed to amplify a 764-bp fragment encoding for a single copy gene BDNF (brain-derived neurotrophic factor).

Sense primer sequence: 5 -AUG GAG AUC UCU GGA TCC ATG ACC ATC CTT TTC CTT-3
Antisense primer sequence: 5 -ACG CGU ACU AGU GGA TCC CTA TCT TCC CCT TTT AAT-3

Quality Control

The Certificate of Analysis (CofA) provides detailed quality control information for each product. The CofA is available here, and is searchable by product lot number, which is printed on each box.

Protocol

The following protocol is optimized for the control DNA and the primers provided with this kit. This protocol may serve as a starting point for any PCR amplification. Critical parameters to optimize include incubation times and temperatures, concentration of Taq DNA Polymerase, primers, MgCl2, and template DNA (2). Since PCR is a powerful technique capable of amplifying trace amounts of DNA, all appropriate precautions should be taken to avoid cross-contamination. Amplification reactions should be assembled in a DNA-free environment. Use of "clean" dedicated automatic pipettors and aerosol resistant barrier tips are recommended. Always keep the control DNA and other templates to be amplified isolated from the other components. PCR products should be analyzed in an area separate from the reaction assembly area.

  1. Add the following components to a sterile 0.5-mL microcentrifuge tube on ice:

    Components Volume (μL)
     Final Concentration
    Autoclaved distilled water 78.5---
    10X PCR buffer minus Mg*10 1X
    50 mM MgCl2*
    3 1.5 mM
    10 mM dNTP mixture2 0.2 mM each
    Primer mix (10 μM each)
     5
    0.5 μM each
    Control DNA (200 ng/μL)
    1
    200 ng (6 × 104 molecules)
    Taq DNA Polymerase (5 U/μL)
     0.5
    2.5 units

     Total Volume
     100 μL

    If desired, a master mix of buffer, MgCl2, dNTP's and Taq DNA polymerase can be prepared for multiple reactions. This minimizes reagent loss and enables accurate pipetting.

    *Note: 10X PCR Buffer plus Mg can be used instead of the separate 10X PCR buffer minus Mg and 50 mM MgCl2 to achieve the same final concentration.

  2. Cap the tubes and centrifuge briefly to collect the contents to the bottom of the tube.

  3. Incubate the tubes in a thermocycler at 94° C for 3 min to completely denature the template.

  4. Perform 35 cycles of PCR amplification at:

    a. Denature: 94° C for 45 s
    b. Anneal: 55° C for 30 s
    c. Extend: 72° C for 1 min 30 s

  5. Incubate for an additional 10 min at 72° C and maintain the reaction at 4° C. The samples can be stored at –20  C until use.

  6. Analyze the amplification products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.

"Hot-Start" Protocol

In the "Hot-Start" method (4), the addition of Taq DNA Polymerase is withheld until the template is completely denatured, so as to ensure high specificity of the products being synthesized.

  1. Add all the components as in Basic Protocol, except Taq DNA Polymerase.

  2. Cap the tubes and centrifuge briefly to collect the contents to the bottom of the tube.

  3. Incubate the tubes in a thermal cycler at 94° C for 3 min to completely denature the template.

  4. After denaturation at 94° C, maintain the reaction at 80° C.

  5. Add 0.5 μL of Taq DNA Polymerase (2.5 U) to each reaction. Be certain to add the enzyme beneath the layer of silicone oil.

  6. Continue with 35 cycles of denaturation annealing and extension as in Basic Protocol.

References

  1. Saiki, R.K. et al. (1985) Science 230, 1350.

  2. Innis, M.A., Gelfand, D.H., Sninsky, J.J., and White, T.J., eds. (1990) PCR Protocols: A Guide to Methods and Applications, Academic Press, San Diego, CA.

  3. Scharf, S.J. et al. (1986) Science 233, 1076.

  4. D'Aquilla, R.T. et al. (1991) Nucleic Acids Res. 19, 3749.
MAN0000791           18-Jun-2010