ReproFast proofreading Polymerase

Technical Data:

Specifications:
5 U/µL
3' - 5' Exonuclease activity (proof-reading activity)

application:

Polymerase mixture to be used for long fragments in PCR

Unit Definition:

One unit is defined as the amount of enzyme which will convert 10 nmoles of dNTPs to an acid-insoluble form in 30 min at 72°C under the assay conditions (25 mM TAPS (tris-(hydroxymethyl)-methyl-amino-propanesulfonic acid, sodium salt) pH 9.3 (25°C), 50 mM

Source

recombinant

   MSDS

   Protocols

   Manuals

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).

Primers

• 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.

Hier finden Sie Artikel und Literaturzitate, in denen die Autoren auf die hohe Qualität dieses Genaxxonprodukts vertrauen.
Listed below are articles and references, in which the authors trust in the high quality of this Genaxxon product.

Quelle/Source: NCBI PubMed >

MpsAB is important for Staphylococcus aureus virulence and growth at atmospheric CO₂ levels
Sook-Ha Fan, Patrick Ebner, Sebastian Reichert, Tobias Hertlein, Susanne Zabel, Aditya Kumar Lankapalli, Kay Nieselt, Knut Ohlsen, Friedrich Götz
Nat Commun. 2019; 10: 3627. Published online 2019 Aug 9. doi: 10.1038/s41467-019-11547-5
PMCID: PMC6689103

Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis
Frank R. Bengelsdorf, Anja Poehlein, Sonja Linder, Catarina Erz, Tim Hummel, Sabrina Hoffmeister, Rolf Daniel, Peter Dürre
Front Microbiol. 2016; 7: 1036.  Published online 2016 Jul 7. doi: 10.3389/fmicb.2016.01036
PMCID: PMC4935695

A Putative Non-Canonical Ras-Like GTPase from P. falciparum: Chemical Properties and Characterization of the Protein
Annette Kaiser, Barbara Langer, Jude Przyborski, David Kersting, Mirko Krüger
PLoS One. 2015; 10(11): e0140994.  Published online 2015 Nov 5. doi: 10.1371/journal.pone.0140994
PMCID: PMC4634863

Functionally redundant but dissimilar microbial communities within biogas reactors treating maize silage in co-fermentation with sugar beet silage
Susanne G Langer, Sharif Ahmed, Daniel Einfalt, Frank R Bengelsdorf, Marian Kazda
Microb Biotechnol. 2015 Sep; 8(5): 828–836.  Published online 2015 Jul 22. doi: 10.1111/1751-7915.12308
PMCID: PMC4554470

Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors
Christina Gabris, Frank R Bengelsdorf, Peter Dürre
Microb Biotechnol. 2015 Sep; 8(5): 865–873.  Published online 2015 Jun 18. doi: 10.1111/1751-7915.12299
PMCID: PMC4554474

Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions
Steffi Noack-Schönmann, Tanja Bus, Ronald Banasiak, Nicole Knabe, William J Broughton, H Den Dulk-Ras, Paul JJ Hooykaas, Anna A Gorbushina
AMB Express. 2014; 4: 80.  Published online 2014 Nov 4. doi: 10.1186/s13568-014-0080-5
PMCID: PMC4230810

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors
Jennifer Mehlhorn, Helena Steinocher, Sebastian Beck, John T. M. Kennis, Peter Hegemann, Tilo Mathes
PLoS One. 2013; 8(11): e79006.  Published online 2013 Nov 1. doi: 10.1371/journal.pone.0079006
PMCID: PMC3815312

Liver hyperplasia after tamoxifen induction of Myc in a transgenic medaka model
Luciana A. Menescal, Cornelia Schmidt, Daniel Liedtke, Manfred Schartl
Dis Model Mech. 2012 Jul; 5(4): 492–502.  Published online 2012 Mar 15. doi: 10.1242/dmm.008730
PMCID: PMC3380712

Gene Cluster Involved in the Biosynthesis of Griseobactin, a Catechol-Peptide Siderophore of Streptomyces sp. ATCC 700974
Silke I. Patzer, Volkmar Braun
J Bacteriol. 2010 Jan; 192(2): 426–435.  Published online 2009 Nov 13. doi: 10.1128/JB.01250-09
PMCID: PMC2805312

Replacement of a Phenylalanine by a Tyrosine in the Active Site Confers Fructose-6-phosphate Aldolase Activity to the Transaldolase of Escherichia coli and Human Origin
Sarah Schneider, Tatyana Sandalova, Gunter Schneider, Georg A. Sprenger, Anne K. Samland
J Biol Chem. 2008 Oct 31; 283(44): 30064–30072.  doi: 10.1074/jbc.M803184200
PMCID: PMC2662071