Taq DNA Polymerase S (high specificity)
Advantages at a glance
- stable at RT (+15 to +30°C) for at least 2 weeks
- high specifity amplification
- processes up to >7kb
- extension rate: 2-4kb/min at 72°C
- extra addition of A
Ready to ship today,
Delivery time 1-2 workdays
Shipment: on wet ice. Store at -20°C. For laboratory usage only!
High-quality Taq DNA Polymerase from Genaxxon is a highly processive 5' - 3' DNA Polymerase, lacking 3' - 5' exonuclease activity. The high processivity and fidelity of Genaxxon bioscience Taq Polymerase allows amplification of DNA fragment of up to >7 kb. Genaxxon bioscience Taq Polymerase is delivered with 10X reaction buffer and separate MgCL2.The enzyme is delivered with our buffer component 'Buffer-S'. The buffer is optimised for high specificity amplification of DNA-templates. Our complete buffer contains 15mM MgCl2.
Free Taq DNA Polymerase test sample available!No shipping costs within Germany.
With our high quality dNTPs as Set (M3015.4100 and M3015.0250) or Mix (M3016.1010) or our DNA Ladders and our favourable standard agarose (M3044) we can offer additional high quality products for your PCR.
Product Specifications
Concentration: 5 units/µL
Substrate analogs: dNTP, ddNTP, fluorescent dNTP/ddNTP
Extension rate: 2-4 kb/min. at 72°C
Half-life: 20min. at 95°C, 60min. at 94°C
5’-3’ exonuclease activity: Yes
Extra addition of A: Yes
3’-5’ exonuclease activity: No
Nuclease contamination: No
Protease contamination: No
RNase contamination: No
Self-priming activity: No
Sicherheits Hinweise / Safety
Klassifizierungen / Classification
eclass-Nr: 32-16-05-02
Documents:
Safety Data SheetProtocols
Certificate
Manuals
Category List
Source: NCBI PubMed
Detection and molecular identification of Blastocystis isolates from humans and cattle in northern Egypt.
Sarah Mohamed Abdo, Hosny El-Adawy, Hoda Fahmy Farag, Hend Aly El-Taweel, Heba Elhadad, Ayman Abdel-Moamen El-Badry
J Parasit Dis. 2021 Sep;45(3):738-745.
doi: 10.1007/s12639-021-01354-5.
PMCID: PMC8368324.
Polyphasic study of antibiotic-resistant enterobacteria isolated from fresh produce in Germany and description of Enterobacter vonholyi sp. nov. isolated from marjoram and Enterobacter dykesii sp. nov. isolated from mung bean sprout.
Gyu-Sung Cho, Maria Stein, Gregor Fiedler, Etinosa O. Igbinosa, Linnéa Philine Koll, Erik Brinks, Jana Rathje, Horst Neve, Charles M A P Franz
Syst Appl Microbiol. 2021 Jan;44(1):126174.
doi: 10.1016/j.syapm.2020.126174.
PMID: 33370657.
Chip-based duplex real-time PCR for water quality monitoring concerning Legionella pneumophila and Legionella spp.
Cornelia Reuter, Stefanie Hentschel, Antje Breitenstein, Eileen Heinrich, Oliver Aehlig, Thomas Henkel, Andrea Csáki, Wolfgang Fritzsche
Water Environ. J. 2020, 35: 371-380.
doi: 10.1111/wej.12635
Coding and noncoding somatic mutations in candidate genes in basal cell carcinoma.
Maria Giovanna Maturo, Sivaramakrishna Rachakonda, Barbara Heidenreich, Cristina Pellegrini, Nalini Srinivas, Celia Requena, Carlos Serra-Guillen, Beatriz Llombart, Onofre Sanmartin, Carlos Guillen, Lucia Di Nardo, Ketty Peris, Maria Concetta Fargnoli, Eduardo Nagore, Rajiv Kumar
Sci Rep. 2020 May 14;10(1):8005.
doi: 10.1038/s41598-020-65057-2.
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Identification, Genotyping and Antimicrobial Susceptibility Testing of Brucella spp. Isolated from Livestock in Egypt
Aman Ullah Khan, Waleed S. Shell, Falk Melzer, Ashraf E. Sayour, Eman Shawkat Ramadan, Mandy C. Elschner, Amira A. Moawad, Uwe Roesler, Heinrich Neubauer, Hosny El-Adawy
Microorganisms. 2019 Dec; 7(12): 603.
doi: 10.3390/microorganisms7120603
PMCID: PMC6955977
Metabolic Engineering of Escherichia coli for para-Amino-Phenylethanol and para-Amino-Phenylacetic Acid Biosynthesis
Behrouz Mohammadi Nargesi, Georg A. Sprenger, Jung-Won Youn
Front Bioeng Biotechnol. 2018; 6: 201.
doi: 10.3389/fbioe.2018.00201
PMCID: PMC6328984
Genome-wide polyadenylation site mapping datasets in the rice blast fungus Magnaporthe oryzae
Marco Marconi, Ane Sesma, Julio Luis Rodríguez-Romero, María Lourdes Rosano González, Mark D. Wilkinson
Sci Data. 2018; 5: 180271.
doi: 10.1038/sdata.2018.271
PMCID: PMC6257040
Blasticidin-S deaminase, a new selection marker for genetic transformation of the diatom Phaeodactylum tricornutum
Jochen M. Buck, Carolina Río Bártulos, Ansgar Gruber, Peter G. Kroth
PeerJ. 2018; 6: e5884.
doi: 10.7717/peerj.5884
PMCID: PMC6250098
Deciphering the Adaptation of Corynebacterium glutamicum in Transition from Aerobiosis via Microaerobiosis to Anaerobiosis
Julian Lange, Eugenia Münch, Jan Müller, Tobias Busche, Jörn Kalinowski, Ralf Takors, Bastian Blombach
Genes (Basel) 2018 Jun; 9(6): 297.
doi: 10.3390/genes9060297
PMCID: PMC6027265
Identification, differentiation and antibiotic susceptibility of Gallibacterium isolates from diseased poultry
Hosny El-Adawy, Herbert Bocklisch, Heinrich Neubauer, Hafez Mohamed Hafez, Helmut Hotzel
Ir Vet J. 2018; 71: 5.
doi: 10.1186/s13620-018-0116-2
PMCID: PMC5799919
An application of competitive reporter monitored amplification (CMA) for rapid detection of single nucleotide polymorphisms (SNPs)
Juliane Havlicek, Eric Rivera-Milla, Peter Slickers, Sönke Andres, Silke Feuerriegel, Stefan Niemann, Matthias Merker, Ines Labugger
PLoS One. 2017; 12(8): e0183561.
doi: 10.1371/journal.pone.0183561
PMCID: PMC5574540
The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome
Coral González-Prieto, Richard Gabriel, Christoph Dehio, Manfred Schmidt, Matxalen Llosa
Appl Environ Microbiol. 2017 Jun 15; 83(12): e00207-17.
doi: 10.1128/AEM.00207-17
PMCID: PMC5452801
Genetic alterations in seborrheic keratoses
Barbara Heidenreich, Evygenia Denisova, Sivaramakrishna Rachakonda, Onofre Sanmartin, Timo Dereani, Ismail Hosen, Eduardo Nagore, Rajiv Kumar
Oncotarget. 2017 May 30; 8(22): 36639–36649.
doi: 10.18632/oncotarget.16698
PMCID: PMC5482683
Epidemiological tracing of bovine tuberculosis in Switzerland, multilocus variable number of tandem repeat analysis of Mycobacterium bovis and Mycobacterium caprae.
Giovanni Ghielmetti, Simone Scherrer, Ute Friedel, Daniel Frei, Dominique Suter, Lukas Perler, Max M Wittenbrink
PLoS One. 2017 Feb 21;12(2):e0172474.
doi: 10.1371/journal.pone.0172474.
PMCID: PMC5319696.
Post-translational Serine/Threonine Phosphorylation and Lysine Acetylation: A Novel Regulatory Aspect of the Global Nitrogen Response Regulator GlnR in S. coelicolor M145
Rafat Amin, Mirita Franz-Wachtel, Yvonne Tiffert, Martin Heberer, Mohamed Meky, Yousra Ahmed, Arne Matthews, Sergii Krysenko, Marco Jakobi, Markus Hinder, Jane Moore, Nicole Okoniewski, Boris Maček, Wolfgang Wohlleben, Agnieszka Bera
Front Mol Biosci. 2016; 3: 38.
doi: 10.3389/fmolb.2016.00038
PMCID: PMC4977719
Quantification of Slackia and Eggerthella spp. in Human Feces and Adhesion of Representatives Strains to Caco-2 Cells
Gyu-Sung Cho, Felix Ritzmann, Marie Eckstein, Melanie Huch, Karlis Briviba, Diana Behsnilian, Horst Neve, Charles M. A. P. Franz
Front Microbiol. 2016; 7: 658.
doi: 10.3389/fmicb.2016.00658
PMCID: PMC4860493
Transcriptional Regulation of the β-Type Carbonic Anhydrase Gene bca by RamA in Corynebacterium glutamicum
Adnan Shah, Bernhard J. Eikmanns
PLoS One. 2016; 11(4): e0154382.
doi: 10.1371/journal.pone.0154382
PMCID: PMC4847777
Mapping of deletion breakpoints at the CDKN2A locus in melanoma: detection of MTAP-ANRIL fusion transcripts
Huaping Xie, P. Sivaramakrishna Rachakonda, Barbara Heidenreich, Eduardo Nagore, Antje Sucker, Kari Hemminki, Dirk Schadendorf, Rajiv Kumar
Oncotarget. 2016 Mar 29; 7(13): 16490–16504.
doi: 10.18632/oncotarget.7503
PMCID: PMC4941330
DNase-Sensitive and -Resistant Modes of Biofilm Formation by Listeria monocytogenes
Marion Zetzmann, Mira Okshevsky, Jasmin Endres, Anne Sedlag, Nelly Caccia, Marc Auchter, Mark S. Waidmann, Mickaël Desvaux, Rikke L. Meyer, Christian U. Riedel
Front Microbiol. 2015; 6: 1428.
doi: 10.3389/fmicb.2015.01428
PMCID: PMC4686886
Skin-Derived C-Terminal Filaggrin-2 Fragments Are Pseudomonas aeruginosa-Directed Antimicrobials Targeting Bacterial Replication
Britta Hansmann, Jens-Michael Schröder, Ulrich Gerstel
PLoS Pathog. 2015 Sep; 11(9): e1005159.
doi: 10.1371/journal.ppat.1005159
PMCID: PMC4570713
Tight Junction Protein 1a regulates pigment cell organisation during zebrafish colour patterning
Andrey Fadeev, Jana Krauss, Hans Georg Frohnhöfer, Uwe Irion, Christiane Nüsslein-Volhard
eLife. 2015; 4: e06545.
doi: 10.7554/eLife.06545
PMCID: PMC4446668
Down-Regulation of ZmEXPB6 (Zea mays β-Expansin 6) Protein Is Correlated with Salt-mediated Growth Reduction in the Leaves of Z. mays L.
Christoph-Martin Geilfus, Dietrich Ober, Lutz A. Eichacker, Karl Hermann Mühling, Christian Zörb
J Biol Chem. 2015 May 1; 290(18): 11235–11245.
doi: 10.1074/jbc.M114.619718
PMCID: PMC4416831
Phylogeny and Differentiation of Reptilian and Amphibian Ranaviruses Detected in Europe
Anke C. Stöhr, Alberto López-Bueno, Silvia Blahak, Maria F. Caeiro, Gonçalo M. Rosa, António Pedro Alves de Matos, An Martel, Alí Alejo, Rachel E. Marschang
PLoS One. 2015; 10(2): e0118633.
doi: 10.1371/journal.pone.0118633
PMCID: PMC4338083
The Staphylococcus aureus NuoL-Like Protein MpsA Contributes to the Generation of Membrane Potential
Sonja Mayer, Wojtek Steffen, Julia Steuber, Friedrich Götz
J Bacteriol. 2015 Mar; 197(5): 794–806.
doi: 10.1128/JB.02127-14
PMCID: PMC4325100
Transcriptional landscape and essential genes of Neisseria gonorrhoeae
Christian W. Remmele, Yibo Xian, Marco Albrecht, Michaela Faulstich, Martin Fraunholz, Elisabeth Heinrichs, Marcus T. Dittrich, Tobias Müller, Richard Reinhardt, Thomas Rudel
Nucleic Acids Res. 2014 Sep 15; 42(16): 10579–10595.
doi: 10.1093/nar/gku762
PMCID: PMC4176332
Molecular evidence for convergent evolution and allopolyploid speciation within the Physcomitrium-Physcomitrella species complex
Anna K Beike, Mark von Stackelberg, Mareike Schallenberg-Rüdinger, Sebastian T Hanke, Marie Follo, Dietmar Quandt, Stuart F McDaniel, Ralf Reski, Benito C Tan, Stefan A Rensing
BMC Evol Biol. 2014; 14: 158.
doi: 10.1186/1471-2148-14-158
PMCID: PMC4227049
Improvement of L-phenylalanine production from glycerol by recombinant Escherichia coli strains: The role of extra copies of glpK, glpX, and tktA genes
Katrin Gottlieb, Christoph Albermann, Georg A Sprenger
Microb Cell Fact. 2014; 13: 96.
doi: 10.1186/s12934-014-0096-1
PMCID: PMC4227036
Molecular Typing of MRSA and of Clinical Staphylococcus aureus Isolates from Iaşi, Romania
Stefan Monecke, Elke Müller, Olivia Simona Dorneanu, Teodora Vremeră, Ralf Ehrich
PLoS One. 2014; 9(5): e97833.
doi: 10.1371/journal.pone.0097833
PMCID: PMC4028265
Bioelectric Signaling Regulates Size in Zebrafish Fins
Simon Perathoner, Jacob M. Daane, Ulrike Henrion, Guiscard Seebohm, Charles W. Higdon, Stephen L. Johnson, Christiane Nüsslein-Volhard, Matthew P. Harris
PLoS Genet. 2014 Jan; 10(1): e1004080.
doi: 10.1371/journal.pgen.1004080
PMCID: PMC3894163
The PHOTOSYNTHESIS AFFECTED MUTANT68–LIKE Protein Evolved from a PSII Assembly Factor to Mediate Assembly of the Chloroplast NAD(P)H Dehydrogenase Complex in Arabidopsis
Ute Armbruster, Thilo Rühle, Renate Kreller, Christoph Strotbek, Jessica Zühlke, Luca Tadini, Thomas Blunder, Alexander P. Hertle, Yafei Qi, Birgit Rengstl, Jörg Nickelsen, Wolfgang Frank, Dario Leister
Plant Cell. 2013 Oct; 25(10): 3926–3943.
doi: 10.1105/tpc.113.114785
PMCID: PMC3877787
Variants at the 9p21 locus and melanoma risk
Livia Maccioni, Panduranga Sivaramakrishna Rachakonda, Justo Lorenzo Bermejo, Dolores Planelles, Celia Requena, Kari Hemminki, Eduardo Nagore, Rajiv Kumar
BMC Cancer. 2013; 13: 325.
doi: 10.1186/1471-2407-13-325
PMCID: PMC3702420
Phylogenetic and Molecular Analysis of Food-Borne Shiga Toxin-Producing Escherichia coli
Elisabeth Hauser, Alexander Mellmann, Torsten Semmler, Helen Stoeber, Lothar H. Wieler, Helge Karch, Nikole Kuebler, Angelika Fruth, Dag Harmsen, Thomas Weniger, Erhard Tietze, Herbert Schmidt
Appl Environ Microbiol. 2013 Apr; 79(8): 2731–2740.
doi: 10.1128/AEM.03552-12
PMCID: PMC3623172
German Francisella tularensis isolates from European brown hares (Lepus europaeus) reveal genetic and phenotypic diversity
Wolfgang Müller, Helmut Hotzel, Peter Otto, Axel Karger, Barbara Bettin, Herbert Bocklisch, Silke Braune, Ulrich Eskens, Stefan Hörmansdorfer, Regina Konrad, Anne Nesseler, Martin Peters, Martin Runge, Gernot Schmoock, Bernd-Andreas Schwarz, Reinhard Sting, Kerstin Myrtennäs, Edvin Karlsson, Mats Forsman, Herbert Tomaso
BMC Microbiol. 2013; 13: 61.
doi: 10.1186/1471-2180-13-61
PMCID: PMC3663675
Increased detection rates of EGFR and KRAS mutations in NSCLC specimens with low tumour cell content by 454 deep sequencing
Evgeny A. Moskalev, Robert Stöhr, Ralf Rieker, Simone Hebele, Florian Fuchs, Horia Sirbu, Sergey E. Mastitsky, Carsten Boltze, Helmut König, Abbas Agaimy, Arndt Hartmann, Florian Haller
Virchows Arch. 2013 Apr; 462(4): 409–419.
doi: 10.1007/s00428-013-1376-6
PMCID: PMC3624006
An efficient method for genome-wide polyadenylation site mapping and RNA quantification
Stefan Wilkening, Vicent Pelechano, Aino I. Järvelin, Manu M. Tekkedil, Simon Anders, Vladimir Benes, Lars M. Steinmetz
Nucleic Acids Res. 2013 Mar; 41(5): e65.
doi: 10.1093/nar/gks1249
Correction in: Nucleic Acids Res. 2013 Jul; 41(12): 6370.
PMCID: PMC3597643
Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum
Christopher F. Schuster, Jung-Ho Park, Marcel Prax, Alexander Herbig, Kay Nieselt, Ralf Rosenstein, Masayori Inouye, Ralph Bertram
J Bacteriol. 2013 Jan; 195(1): 115–125.
doi: 10.1128/JB.00400-12
PMCID: PMC3536171
Detection of Staphylococcal Cassette Chromosome mec Type XI Carrying Highly Divergent mecA, mecI, mecR1, blaZ, and ccr Genes in Human Clinical Isolates of Clonal Complex 130 Methicillin-Resistant Staphylococcus aureus
Anna C. Shore, Emily C. Deasy, Peter Slickers, Grainne Brennan, Brian O'Connell, Stefan Monecke, Ralf Ehricht, David C. Coleman
Antimicrob Agents Chemother. 2011 Aug; 55(8): 3765–3773.
doi: 10.1128/AAC.00187-11
PMCID: PMC3147645
Quantitative Trait Loci Involved in Sex Determination and Body Growth in the Gilthead Sea Bream (Sparus aurata L.) through Targeted Genome Scan
Dimitrios Loukovitis, Elena Sarropoulou, Costas S. Tsigenopoulos, Costas Batargias, Antonios Magoulas, Apostolos P. Apostolidis, Dimitrios Chatziplis, Georgios Kotoulas
PLoS One. 2011; 6(1): e16599.
doi: 10.1371/journal.pone.0016599
PMCID: PMC3031595
Extreme RNA editing in coding islands and abundant microsatellites in repeat sequences of Selaginella moellendorffii mitochondria: the root of frequent plant mtDNA recombination in early tracheophytes.
Julia Hecht, Felix Grewe, Volker Knoop
Genome Biol Evol. 2011;3:344-58.
doi: 10.1093/gbe/evr027.
PMCID: PMC5654404.
The Apparent Malate Synthase Activity of Rhodobacter sphaeroides Is Due to Two Paralogous Enzymes, (3S)-Malyl-Coenzyme A (CoA)/β-Methylmalyl-CoA Lyase and (3S)- Malyl-CoA Thioesterase
Tobias J. Erb, Lena Frerichs-Revermann, Georg Fuchs, Birgit E. Alber
J Bacteriol. 2010 Mar; 192(5): 1249–1258.
doi: 10.1128/JB.01267-09
PMCID: PMC2820834
Highlights of the Didymellaceae: A polyphasic approach to characterise Phoma and related pleosporalean genera
M.M. Aveskamp, J. de Gruyter, J.H.C. Woudenberg, G.J.M. Verkley, P.W. Crous
Stud Mycol. 2010; 65: 1–60.
doi: 10.3114/sim.2010.65.01
PMCID: PMC2836210
Molecular Analysis of Virulence Profiles and Shiga Toxin Genes in Food-Borne Shiga Toxin-Producing Escherichia coli
T. Slanec, A. Fruth, K. Creuzburg, H. Schmidt
Appl Environ Microbiol. 2009 Oct; 75(19): 6187–6197.
doi: 10.1128/AEM.00874-09
PMCID: PMC2753087
A trans-splicing group I intron and tRNA-hyperediting in the mitochondrial genome of the lycophyte Isoetes engelmannii
Felix Grewe, Prisca Viehoever, Bernd Weisshaar, Volker Knoop
Nucleic Acids Res. 2009 Aug; 37(15): 5093–5104.
doi: 10.1093/nar/gkp532
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A Microsatellite-Based Genetic Linkage Map of the Cichlid Fish, Astatotilapia burtoni (Teleostei): A Comparison of Genomic Architectures Among Rapidly Speciating Cichlids
Matthias Sanetra, Frederico Henning, Shoji Fukamachi, Axel Meyer
Genetics. 2009 May; 182(1): 387–397.
doi: 10.1534/genetics.108.089367
PMCID: PMC2674835
Multiple Didymella teleomorphs are linked to the Phoma clematidina morphotype
J.H.C. Woudenberg, M.M. Aveskamp, J. de Gruyter, A.G. Spiers, P.W. Crous
Persoonia. 2009 Jun; 22: 56–62.
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Sexual Reproduction as the Cause of Heat Resistance in the Food Spoilage Fungus Byssochlamys spectabilis (Anamorph Paecilomyces variotii)
Jos Houbraken, János Varga, Emilia Rico-Munoz, Shawn Johnson, Robert A. Samson
Appl Environ Microbiol. 2008 Mar; 74(5): 1613–1619.
doi: 10.1128/AEM.01761-07
PMCID: PMC2258620
Molecular Characterization and Distribution of Genes Encoding Members of the Type III Effector NleA Family among Pathogenic Escherichia coli Strains
Kristina Creuzburg, Herbert Schmidt
J Clin Microbiol. 2007 Aug; 45(8): 2498–2507.
doi: 10.1128/JCM.00038-07
PMCID: PMC1951211
Nuclear Factor I X Deficiency Causes Brain Malformation and Severe Skeletal Defects
Katrin Driller, Axel Pagenstecher, Markus Uhl, Heymut Omran, Ansgar Berlis, Albert Gründer, Albrecht E. Sippel
Mol Cell Biol. 2007 May; 27(10): 3855–3867.
doi: 10.1128/MCB.02293-06
PMCID: PMC1899988
Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution
Kristian M. Müller, Sabine C. Stebel, Susanne Knall, Gregor Zipf, Hubert S. Bernauer, Katja M. Arndt
Nucleic Acids Res. 2005; 33(13): e117.
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For every template/primer pair, the optimal reaction conditions have to be evaluated empirically, changing the primer/template
ratio, the ionic strength (with MgSO4) and the cycle parameters (time and temperatures).
DNA Template
Amplification of templates with high GC content, strong secondary structure, low concentrations, or which produce products greater than 5 kb, may require adaptation of the following parameters:
- Use high quality, purified DNA templates.
- Approximately 10E4 copies of target DNA are required to detect product in 25-30 PCR cycles.
- Use 1pg–1ng of plasmid or viral templates.
- Use 1ng–1µg of genomic templates.
- Higher DNA concentrations decrease amplicon specificity (i.e., extra bands are more likely), particularly when a large number of cycles are employed.
- Use the higher DNA concentrations when fewer cycles are desired (e.g. to increase fidelity).
- Generally 20-30 nucleotides in length.
- Ideal GC content is 40-60%.
- Space GC residues evenly within the primer.
- Primer pairs should have Tms within 5°C of each other.
- Avoid secondary structure (i.e., hairpins) within each primer and potential dimerization between the primers present.
- When engineering sites into the end of primers, 4-6 extra bases should be added 5´ to the site.
- Final concentration should be 0.05-1 µM, typically 0.1-0.5 µM of each primer.
- Higher concentrations may increase secondary priming and create spurious amplification products.
Magnesium Concentration
- 1.5-2.0 mM is optimal for Taq DNA Polymerase, but the ideal concentration depends on template, buffer, DNA and dNTPs (each has the potential to chelate magnesium).
- If [Mg2+] is too low, no PCR product will be seen.
- If [Mg2+] is too high, undesired PCR products may be seen.
Deoxyribonucleotide triphosphates (dNTPs) >
- Typical concentration is 200 µM of each dNTP.
- 50-100 µM enhances fidelity of polymerization, but reduces yields.
- Higher concentrations increase yields particularly in long PCR, but can reduce fidelity.
DNA Polymerase
- The choice of the correct polymerase depends among other things on the purpose as well as on the template used (standard PCR: Taq DNA Polymerase S > with high accuracy, Taq Polymerase E > with high yield; master mixes: Standard PCR master mix > or RedMasterMix > with red dye).
- For multiplex PCR, there exist special multiplex master mixes >.
- Hot start applications are recommended to increase specificity or if you use difficult templates. For this purpose there exist special Hot Start Polymerases >.
- For PCRs for the purpose of cloning or other procedures requiring a low error rate, there are thermostable high fidelity proofing polymerases such as Pfunds >, ExactRun >, ReproFast >, or ReproHot (KOD) Proofreading Polymerase >. These enzymes make far fewer errors during amplification and increase the chances of an amplicon without mistakes.
- For genotyping and other applications where a high discrimination rate is required, there is a new highly selective DNA polymerase, SNP Pol DNA Polymerase >. It specifically distinguishes mismatched primer-template complexes and ony produces specific amplicons in case of perfectly matched primer pairs.
- For the real-time quantitative PCR > there exist highly specialized qPCR master mixes. Hereby, the choice of the correct master mix depends on the qPCR device you are using. It is important to determine whether and how much ROX should be contained in the qPCR master mix and whether it should be a qPCR green master mix with green fluorescent dye > or a qPCR probe master mix > without a fluorescent dye. A new lyophilized qPCR probe master mix in beads format, which is stable at room temperature, is available from Genaxxon (LyoBalls >).
- The amount of DNA polymerase used in the PCR reaction can significantly influence the PCR result (use 1.25-1.5 units Taq Polymerase > for a 50μL volume).
General Guidelines
Annealing Temperature and Duration
- Match the Tms within 5°C of each other.
- Typical annealing temperatures are 5°C below the lowest primer's Tm and often fall in the range of 50-60°C.
- Test higher annealing temperatures if spurious amplification products are observed.
- Typical annealing times are 15-30 seconds.
Extension Time
- Extensions are normally performed at 68°C.
- In general, use extension times of one minute per 1000 base (1 kb) pairs (e.g. 3 minutes for a 3 kb product).
- For products less than 1 kb, use 45-60 seconds.
- Products greater than 3 kb, or reactions using more than 30 cycles, may require longer extensions.
The amplification of templates with high GC content, strong secondary structures, low concentrations or amplicons more than 5 kb often require optimization of the PCR conditions. Typically, 15-30 seconds of denaturation should be performed at 95 ° C during PCR.
