KOD Hot Start DNA Polymerase

Description
KOD Hot Start DNA Polymerase 200 U 1000 U 71086-3 71086-4

KOD Hot Start DNA Polymerase is a premixed complex of KOD HiFi DNA Polymerase and two monoclonal antibodies that inhibit the DNA polymerase and 3'!5' exonuclease activites during PCR reaction assembly at ambient temperatures (1). KOD Hot Start couples the high fidelity, fast extension speed and processivity of KOD HiFi with high specificity of an antibody-mediated hot start. Non-specific amplification is reduced because mispriming events during set up and the initial temperature increase are avoided. This enzyme efficiently amplifies genomic and phage/plasmid DNA targets up to 12 and 20 kbp, respectively. GC-rich targets are also efficiently amplified. KOD Hot Start DNA Polymerase produces blunt-ended DNA products that are suitable for cloning in Novagens Perfectly Blunt and LIC Systems and is compatible with site-directed mutagenesis protocols
Enzyme Species Fidelity* (mutation frequency) Elongation rate (bases/second) Processivity** (nucleotide bases) KOD HiFi DNA Polymerase Thermocccus kodakaraensis 0.0035 106138 > 300 Pfu DNA Polymerase Pyrococcus furiosus 0.0039 25 < 20 Taq DNA Polymerase Thermus aquaticus YT-1 0.013 61 not determined

* Fidelity was measured by the authors as mutation frequency in PCR products using a sensitive blue/white phenotypic assay with a 5.2 kbp lacZ plasmid as template (2). ** Processivity is defined as the number of nucleotides that can be extended in one catalytic reaction by one DNA polymerase molecule.

Unit definition: One unit is defined as the amount of enzyme that will catalyze the incorporation of 10 nmol of dNTP into acid insoluble form in 30 minutes at 75C in a reaction containing 20 mM Tris-HCl (pH 7.5 at 25C), 8 mM MgCl2, 0.5 mM DTT, 50 g/ml BSA, 150 M each of dATP, dCTP, dGTP, dTTP (a mix of unlabeled and [3H]-dTTP) and 150 g/ml activated calf thymus DNA.

2003 Novagen , a brand of EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. Perfectly Blunt, the Novagen logo and Novagen name are trademarks of EMD Biosciences, Inc. KOD Polymerases are manufactured by TOYOBO and distributed by Novagen, Inc. KOD XL DNA Polymerase is licensed under U.S. Patent No. 5,436,149 owned by Takara Shuzo, Co., Ltd. A license under U.S. Patents 4,683,202, 4,683,195 4,965,188 and 5,075,216 or their foreign counterparts, owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd (Roche), has an up-front component and a running-royalty component. The purchase price of this product includes limited, nontransferable rights under the running-royalty component to use only this amount of the product to practice the Polymerase Chain Reaction (PCR) and related processes described in said patents solely for the research and development activities of the purchaser when this product is used in conjunction with a thermal cycler whose use is covered by the up-front fee component. Rights to the up-front fee component must be obtained by the end user in order to have a complete license to use this product in the PCR process. These rights under the up-front fee component may be purchased from Applied Biosystems or obtained by purchasing an Authorized Thermal Cycler. No right to perform or offer commercial services of any kind using PCR, including without limitation reporting the results of purchasers activities for a fee or other commercial consideration, is hereby granted by implication or estoppel. Further information on purchasing licenses to practice the PCR Process may be obtained by contacting the Director of Licensing and Applied Biosystems, 850 Lincoln Drive, Foster City, California 94494 or the Licensing Department at Roche Molecular Systems, Inc. 1145 Atlantic Avenue, Alameda California 94501. All Novagen products are sold for research use only.

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KOD Hot Start DNA Polymerase

Components
KOD Hot Start DNA Polymerase 200 U or 5 200 U KOD Hot Start DNA Polymerase (1 U/l in 50 mM Tris-HCl, 1mM DTT, 0.1 mM EDTA, 50% glycerol, 0.001% Nonidet P-40, 0.001% Tween-20, pH 8.0) 1.2 ml or 5 1.2 ml 10X PCR Buffer for KOD Hot Start DNA Polymerase 1 ml or 5 1 ml 25 mM MgSO4 1 ml or 5 1 ml dNTPs (2 mM each)

Storage
Store all components at 20C.

KOD Hot Start DNA Polymerase

In many cases, the standard reactions described below will provide satisfactory amplification. Remember to include a negative control reaction lacking only template; inclusion of a positive control reaction using a template known to amplify with the primers may also be helpful. Concentrations of enzyme, MgSO4, template and primers can be varied to optimize the reaction.

Standard PCR
1. For each 50 l reaction, assemble the following in a 0.5 ml PCR tube at room temperature or on ice. 30 l 5 l 5 l 2 l 1 l 3 l 3 l 1 l 50 l Note: 2. PCR Grade Water 10X PCR Buffer for KOD Hot Start DNA Polymerase dNTPs (final concentration 0.2 mM) MgSO4 (final concentration 1 mM) template DNA 5' primer (5 pmol/l, final concentration 0.3 M) 3' primer (5 pmol/l, final concentration 0.3 M) KOD Hot Start DNA Polymerase (1 U/l) total volume

Amplification of targets greater than 3 kbp may require more polymerase. Mix gently and centrifuge briefly if necessary to bring reaction components to the bottom of the tube. If necessary, add mineral oil to cover the reaction, cap the tubes, and place in thermal cycler. Activate the polymerase by heating for 2 min at 94C followed by: Denature 15 sec at 94C Anneal 30 sec at 60C Extend 20 sec/kbp at 72C Repeat for 3040 cycles 4. To analyze the reaction products, remove a 10 l sample from beneath the oil overlay and add to appropriate loading buffer. Load and run a 1% agarose gel containing 0.5 g/ml ethidium bromide and visualize the bands under UV illumination.

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KOD Hot Start DNA Polymerase

GC-rich and genomic DNA templates
1. For each 50 l reaction, assemble the following in a 0.5 ml PCR tube at room temperature. 30 l 5 l 5 l 2 l 1 l 3 l 3 l 1 l 50 l Note: PCR Grade Water 10X PCR Buffer for KOD Hot Start DNA Polymerase dNTPs (final concentration 0.2 mM) MgSO4 (final concentration 1 mM) template DNA 5' primer (5 pmol/l, final concentration 0.3 M) 3' primer (5 pmol/l, final concentration 0.3 M) KOD Hot Start DNA Polymerase (1 U/l) total volume

The addition of 25% DMSO (3-4) or 1 M betaine (5-6) can improve amplification and will not decrease the fidelity. 2. Mix gently and centrifuge briefly if necessary to bring reaction components to the bottom of the tube. If necessary, add mineral oil to cover the reaction, cap the tubes and place in thermal cycler. Activate the polymerase by heating for 2 min at 94C followed by: Denature 15 sec at 94C Anneal 30 sec at Primer Tm (510)C Extend 1 min/kbp at 68C Repeat for 2535 cycles 4. To analyze the reaction products, remove a 10 l sample from beneath the oil overlay and add to appropriate loading buffer. Load and run an agarose gel containing 0.5 g/ml ethidium bromide and visualize the bands under UV illumination.

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Additional guidelines
Primers Primer design is important for successful PCR amplification. The primers should be 21 bases long or longer for best results. In addition, the G/C content should be approximately 4060% (a G/C content greater than 60% may require a higher denaturation temperature or a longer denaturation time). Primer pairs should exhibit similar melting temperatures (Tm). Primers for two-step cycling programs should be designed with a high Tm value to ensure proper annealing and extension at the same temperature. In general, use an annealing temperature that is 510C less than the lowest Tm of the primer pair as a starting point. There are several methods for determining the Tm of a primer. The nearest-neighbor method (7) using 50 mM monovalent salt is recommended for accurate Tm prediction. Unlike other methods, the nearest-neighbor method takes into account the primer sequence and other variables such as salt and DNA concentration. The Tm can also be calculated with the % GC method (8). The most general method of calculating the Tm is based on the number of adenine (A), thymidine (T), guanidine (G) or cytosine (C) bases where Tm(C) = 2(NA + NT) + 4(NG + NC.). However, the exact Tm of a given primer may be affected by DNA concentration, presence of denaturants (e.g., DMSO), and nucleotide modifications (e.g., biotin, fluorescent dyes, etc.). Template DNA The suggested amount of template DNA for amplification is 50 ng phage DNA, 1 ng plasmid DNA, 200 ng genomic DNA and 2 l of a 20-l reverse transcriptase reaction mix. Extension The extension temperature may be varied from 6872C. In some cases a 68C could be used to reduce smearing. A longer extension time up to 1 min/kbp may increase yield. Long target DNA Amplification of a long target from plasmid DNA can be improved by increasing the concentration of MgSO4 from 1.0 to 1.5 mM, adding 25% DMSO, adding 1 M betaine, and/or increasing the number of cycles.
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KOD Hot Start DNA Polymerase

Troubleshooting
Symptom
No PCR product

Possible cause
Annealing temperature used is too high

Solution
Because the ionic strength of KOD Hot Start Buffer is low compared to typical DNA polymerase buffers, it is recommended to decrease annealing temperatures by 510C. Use primers longer than 21 bases. Add DMSO to a final concentration of 25%. DMSO does not change enzyme fidelity. Add betaine to a final concentration of 1M. When using plasmid or phage DNA template (up to 21 kbp) the extension time should be 20 sec/kbp at 72C. When using genomic DNA for a template (up 1012 kbp) the extension time should be 1 min/kbp at 68C.

PCR primers are not long enough Low yield High GC content

Suboptimal PCR conditions

Long target DNA

Add DMSO to a final concentration of 25%. DMSO does not change enzyme fidelity. Add betaine to a final concentration of 1M.

Applications
This section lists references for applications with KOD Hot Start DNA Polymerase. Novagen is continuing to evaluate new applications. Please contact Technical Service for the latest information. Application Gene Cloning Reference Watanabe, M., Miyake, K., Yanae, K., Kataoka, Y., Koizumi, S., Endo, T., Ozaki, A. and Iijima, S. (2002) J. Biochem. (Tokyo) 131, 183191. Tabuchi, M., Tanaka, N., Nishida-Kitayama, J., Ohno, H. and Kishi, F. (2002) Mol. Biol. Cell 13, 43714387. Higasa, K. and Hayashi, K. (2002) Nucleic Acids Res. 30, E11.

Mutagenesis Multiplexed SNP genotyping

References
1. Mizuguchi, H., Nakatsuji, M., Fujiwara, S., Takagi, M. and Imanaka, T. (1999) J. Biochem (Tokyo) 126, 762768. 2. Takagi, M., Nishioka, M., Kakihara, H., Kitabayashi, M., Inoue, H., Kawakami, B., Oka, M. and Imanaka, T. (1997) Applied and Environmental Microbiology 63, 45044510. 3. Cheng, S., Fockler, C., Barnes, W. M. and Higuchi, R. (1994) Proc. Natl. Acad. Sci. USA 91, 56955699. 4. Winship, P. R. (1989) Nucleic Acids Res. 17, 1266. 5. Rees, W. A., Yager, T. D., Korte, J. and von Hippel, P. H. (1993) Biochemistry 32, 137144. 6. Henke, W., Herdel, K., Jung, K., Schnorr, D. and Loening, S. A. (1997) Nucleic Acids Res. 25, 39573958. 7. Breslauer, K. J., Frank, R., Blocker, H. and Marky, L. A. (1986) Proc. Natl. Acad. Sci. 83, 37463750. 8. Howley, P. M., Israel, M. A., Law, M. F. and Martin, M. A. (1979) J. Biol. Chem. 254, 48764883.

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TB341 Rev. B 0503!

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