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Introduction

Elongase® Enzyme Mix is suitable for amplification of long as well as short DNA templates. Elongase® consists of a mixture of Taq and Pyrococcus species GB-D thermostable DNA polymerases. An optimized reaction buffer system is also provided which allows amplification of simple (e.g., plasmid and λ DNA) as well as complex genomic targets. Amplification of templates ≤12 kb can be readily obtained with Elongase®. For targets between 12 kb and 20 kb, reaction parameters need to be optimized. Amplification of targets longer than 20 kb is possible, but success is heavily dependent upon template and primer quality as well as careful optimization of reaction conditions.

Amplification using Taq DNA polymerase is generally limited to DNA templates less than 5 kb. This limitation is believed to be largely due to the inability of Taq DNA polymerase, which lacks proofreading (3’ to 5’ exonuclease) activity (1), to correct nucleotide misincorporations and continue primer elongation (2). Amplification systems which are able to amplify long targets have been described (3,4). In addition to the parent DNA polymerase, these systems contain a proofreading enzyme, which edits the nascent strand to allow subsequent polymerization by the parent enzyme. In this manner, Elongase® is able to amplify targets larger than 5 kb.

Component                                            Part No.                                            Amount
Elongase® Enzyme Mix                            Y02377                                                500 μL
5X Buffer A                                              Y02371                                                5 × 1 mL
5X Buffer B                                              Y02372                                                5 × 1 mL

Elongase® Enzyme Mix:

A Taq/Pyrococcus species GB-D DNA polymerase mixture in storage buffer [20 mM Tris-HCl (pH 8.0 at 25°C), 0.1 mM EDTA, 1 mM DTT, stabilizers, and 50% (v/v) glycerol].

Buffer System:

5X Buffer A [300 mM Tris-SO4, (pH 9.1 at 25°C), 90 mM (NH4)2SO4 and 5 mM MgSO4] and 5X Buffer B [300 mM Tris-SO4, (pH 9.1 at 25°C), 90 mM (NH4)2SO4 and 10 mM MgSO4] which can be differentially combined to optimize [Mg2+].

Intended Use

For research use only. Not intended for any animal or human therapeutic or diagnostic use.

Important Parameters to Consider:

Primers:

  • Design primers which will specifically anneal to the target of interest. For complex genomic targets, design primers with a high Tm (>60°C) so stringent primer annealing conditions can be used. It is convenient to design primers which will sufficiently anneal at 68°C so that a 68°C combined annealing and extension step (two-step cycling) can be employed during thermal cycling.
  • For plasmid DNA, λ DNA, or other relatively abundant targets, use each primer at 400 nM. For more complex
    amplification targets with a lower copy number such as genomic DNA, decrease the concentration of each primer to 200 nM. High primer concentrations, in some cases, initiate excessive competitive non-target amplification, especially when genomic DNA is used as target.
  • The activity of Pyrococcus species GB-D DNA polymerase is inhibited in the presence of deoxyuracil-containing DNA. Therefore, use of dUMP-containing oligonucleotides with Elongase® for long amplification is not recommended.


Input template DNA:

  • The quality of the template DNA is important. To amplify a large target, the input DNA must not be excessively sheared, nicked or otherwise damaged.
  • If dilution of the input DNA is required, be sure to use sterile water as the diluent and not buffer. Carryover of
    experimental sample buffer components along with the DNA may affect the amplification reaction with respect to [Mg2+], primer concentration and enzyme amount and, therefore, reactions may require re-optimization.

Reactions:

  • Keep all components, reaction mixes and samples on ice. After preparation of the samples, transfer them to a pre-heated thermal cycler (94°C) and immediately start the amplification program.
  • For most targets, 1 μL of enzyme mixture is sufficient. For smaller targets (less than 12 kb), 2 μL of enzyme mixture can be used to increase amplification yield. For longer targets, 2 μL of Elongase® is not recommended and may result in the appearance of non-specific products.
  • Use ratios of Buffers A and B to determine optimal [Mg2+] for your amplification reactions. It is often useful to titrate Mg2+ concentrations between 1 and 2 mM (see ratio table). In most cases, Mg2+ concentrations between 1.6 and 2.0 mM are appropriate.
  • In most circumstances, avoid using extensive pre-amplification denaturation which is longer or hotter than necessary since it may result in DNA template damage and reduced or no amplification. A temperature of 94°C for 30–60 s is recommended. Longer (3-min) pre-amplification denaturation times may be necessary for supercoiled DNA.

Equipment:

  • Thin-walled amplification tubes, such as Perkin-Elmer GeneAmp® thin-walled reaction tubes, are recommended. Thickwalled tubes decrease amplification efficiency.
  • It is recommended that Elongase® be used in 50-μL amplification reactions with the following thermal cyclers: The DNA Thermal Cycler, the GeneAmp® PCR System 9600 (Perkin-Elmer), and the PTC-100 Thermal Cycler (MJ Research, Inc.). For other thermal cyclers, reaction conditions may have to be optimized.
  • Use aerosol-resistant tips (ART®) and DNA-free pipets for reaction set-up.

 

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Ordering Information

Sku Name Size Price Qty
10480028 Elongase® Enzyme Mix 500 reactions USD 836.00

Protocol

Prepare two separate mixes on ice, one containing template, primers and nucleotides (Mix 1) and another containing buffer and Elongase® (Mix 2). The two mixes can be subsequently combined in an amplification tube on ice or the reaction can be constructed using a “hot start” method.

  1. Add the following to corresponding microcentrifuge tubes placed on ice. Reaction cocktails can be made when multiple reactions are being constructed. It is important to pipette accurately, so use P2 and P10 pipets when dispensing small volumes. Be careful of any extra reagent which may be on the outside of the pipet tip.

    Reaction set-up:

    Volume per 50-μL reaction

    Component
    Genomic DNA Target Other DNA Targets Final Concentration
    Mix 1      
    10 mM dNTP mix 1 μL 1 μL 200 μM each dNTP
    Forward primer, 10 μM 1 μL 2 μL 200 or 400 nM
    Reverse primer 10 μM 1 μL  2 μL 200 or 400 nM
    Template DNA x μL (≥100 ng) x μL (≥25 pg) ---
    Water to 20 μL to 20 μL ---
    Mix 2      
    5X Buffer A

    10 μL total (Buffer A + Buffer B; see table)

     60 mM Tris-SO4 (pH 9.1), 18 mM (NH4)2SO4, with MgSO4 between 1–2 mM
    5X Buffer B      
    Elongase® Enzyme Mix

    1 μL (or 2 μL for targets < 12 kb)

     
    Water

    to 30 μL

     

    Buffer A and B ratio table:

    Use the following amounts of Buffer A and B to achieve the desired Mg2+ concentration. The combined amount of Buffer A + B should equal exactly 10 μL:

    Buffer

    Final [Mg2+] (mM)

      1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0
    A (μL) 10 9 8 7 6 5 4 3 2 1 0
    B (μL) 0 1 2 3 4 5 6 7 8 9 19



  2. Pipette 20 μL of Mix 1 and 30 μL of Mix 2 into an amplification tube placed in ice. Make sure that all components are at the bottom of the amplification tube. Gently mix the tubes and overlay with two drops of the silicone oil provided. Silicone oil may be not be required for some thermal cyclers.

  3. Perform amplification. The following cycling conditions were established using a DNA Thermal Cycler 480 (Perkin-Elmer) and may have to be altered for other thermal cyclers. Remember that cycling conditions may have to be further optimized for difficult sequences and targets over 20 kb. Annealing and extension steps can be separate (three-step cycling), or they can be combined (two-step cycling) if the primers will sufficiently anneal at 68°C. For all low-copy targets, such as singlecopy genomic targets, use 35 cycles of thermal cycling.

    A. For primers with a Tm below 68°C (three-step cycling):

    1. Pre-amplification denaturation:                                         94°C for 30 s, 1 cycle

    2. Thermal cycling: Denaturation;                                          94°C for 30 s                                   
                                                       Annealing; 55–65°C for 30 s                                                                             
                                                       Extension; 68°C for 45–60 s per kb of target                                   ⇒30– &35 cycles


    B. For primers with a Tm equal to, or greater than 68°C (two-step cycling):

    1. Pre-amplification denaturation:                                         94°C for 30 s

    2. Thermal cycling: Denaturation;                                         94°C for 30 s                                        ⇒    30–35 cycles
                                                         Annealing+Extension; 68°C for 45–60 s per kb of target

                                                      
                                                   
  4. Analyze amplification products. Load at least 20% (10 μL) of the amplification sample per gel lane on a 0.4–0.8% TAE agarose gel.


Product Qualification

The Certificate of Analysis provides detailed quality control information for each product. Certificates of Analysis are available here.

 

Troubleshooting

ProblemPossible causePossible solution
No amplification productTemplate DNA concentration is not optimalIncrease or decrease the concentration of template DNA. Use 50–200 ng of genomic DNA for single-copy targets. Use >25 pg for other targets. In some cases, large template amounts reduce amplification efficiency
 DNA is nicked or otherwise damagedCheck quality by agarose gel analysis and replace DNA if necessary.
 Denaturation time and/or temperature is not optimalIncrease or decrease time and/or temperature as necessary. Do not exceed 94°C for denaturation and try to keep denaturation times ≤30 s, except for supercoiled DNA.
 Extension time is too short.Maintain at least 45–60 s per kb of target length. For genomic targets, use 60 s per kb.
 Primer annealing temperature is too high.Decrease the annealing temperature as necessary.
 Cycle number is too low.Increase cycle number.
Electrophoresis of PCR product yields a smear when stained with ethidium bromide.Enzyme amount is too high.Use 1 μL of Elongase® Enzyme Mix.
 Primer annealing temperature is too low.Increase the annealing temperature to increase the specificity of primer annealing.
 Mg2+ concentration is too high.lTitrate the Mg2+ concentration by mixing Buffers A and B (see ratio table)
Multiple distinct bands are obtained by electrophoresis of PCR productPrimer annealing temperature is too low.Increase annealing temperature to increase the specificity of primer annealing
 Amplification samples were not properly kept on ice
during reaction set up.
Keep reactions on ice or use a “hot-start” method.
 Mg2+ concentration is not optimal.Titrate the Mg2+ concentration by mixing Buffers A and B
(see ratio table).
 Primer sequence(s) are
insufficiently specific.
Re-examine primer sequences and redesign.

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References

  1. Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008.

  2. Innis, M.A., Myambo, K.B., Gelfand, D.H. and Brow, M.A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436.

  3. Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216.

  4. Cheng, S., Fockler, C., Barnes, W.M. and Higuchi, R. (1994) Proc. Natl. Acad. Sci. USA 91, 5695.
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MAN0000831        Rev. date:   7-Jun-2010