Phusion Green Hot Start II High-Fidelity DNA Polymerase, #F-537L
Contents
1. PRODUCT INFORMATION Thermo Scientific Phusion Green Hot Start Il High Fidelity DNA Polymerase F 537L 500 U Lot __ Expiry Date _ Store at 20 C www thermoscientific com onebio Ordering information Component F 537S F 537L k 100 U 500 U Phusion Hot Start Il DNA Polymerase 50 pL 250 pL 5X Phusion Green HF Buffer 2x 1 5 mL 6x 1 5 mL 5X Phusion Green GC Buffer 1 5 mL 2x 1 5 mL 50 mM MgCh solution 1 5 mL 2x 1 5 mL 100 DMSO 500 uL 500 uL Reaction buffer 5X Phusion Green HF Buffer and 5X Phusion Green GC Buffer both contain 7 5 mM MgCl Rev 1 1 Introduction Thermo Scientific Phusion Hot Start Il High Fidelity DNA Polymerase offers superior performance for all PCR applications A unique processivity enhancing domain makes this Pyrococcus like proofreading enzyme extremely processive accurate and rapid The error rate of Phusion Hot Start II DNA Polymerase is equal to that of Phusion DNA Polymerase 4 4 x 10 7 in Phusion HF buffer when determined with a modified acl based method It is approximately 50 fold lower than that of Thermus aquaticus DNA polymerase and 6 fold lower than that of Pyrococcus furiosus DNA polymerase Phusion Hot Start II High Fidelity DNA Polymerase is capable of amplifying long amplicons such as the 7 5 kb genomic and 20 kb A DNA used in Thermo Fisher Scientific quality control assays Phusion Hot Start II DNA Polymerase combines the DNA polymerase and a2. e section 5 3 e Titrate template amount e Shorten extension time e Decrease primer see section 5 4 concentration e Reduce enzyme e Design new primers concentration see section 4 1 7 Component specifications 7 1 Phusion Hot Start Il High Fidelity DNA Polymerase F 549 Thermostable Phusion DNA Polymerase is isolated and purified froman E colistrain expressing the cloned Phusion DNA Polymerase gene Phusion DNA Polymerase possesses the following activities 5 3 DNA polymerase activity and 3 5 exonuclease activity The Affibody ligand is isolated and purified from an E coli strain expressing the cloned Affibody encoding gene Storage buffer 20 mM Tris HCl pH 7 4 at 25 C 0 1 mM EDTA 1mM DTT 100 mM KCI stabilizers 200 g mL BSA and 50 glycerol Unit definition One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTPs into a polynucleotide fraction at 74 C in 30 min Enzyme activity is assayed in the following mixture 25 mM TAPS HCI pH 9 3 at 25 C 50 mM KCI 2 mM MgCl 1 mM B mercaptoethanol 0 75 mM activated salmon milt DNA 100 uM dCTP 200 uM each dATP dGTP dTTP 0 4 MBq ml 3H dCTP 7 2 5X Phusion Green HF Buffer F 538 The 5X Phusion HF Buffer contains 7 5 mM MgCl which provides 1 5 mM MgCl in final reaction conditions 7 3 5X Phusion Green GC Buffer F 539 The 5X Phusion GC Buffer contains 7 5 mM MgCle which provides 1 5 mM MgCl in final reaction co
3. ermofisher com North America Latin America amp APAC ts molbio thermofisher com NOTICE TO PURCHASER The purchase price of this product includes a limited non transferable license under U S and foreign patents owned by BIO RAD Laboratories Inc to use this product No other license under these patents is conveyed expressly or by implication to the purchaser by the purchase of this product This product is sold under license from Affibody AB Sweden PRODUCT USE LIMITATION This product is developed designed and sold exclusively for research purposes and in vitro use only The product was not tested for use in diagnostics or for drug development nor is it suitable for administration to humans or animals Please refer to www thermoscientific com onebio for Material Safety Data Sheet of the product 2013 Thermo Fisher Scientific Inc All rights reserved Affibody is a registered trademark of Affibody AB Sweden All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries
4. imer B X uL X uL 0 5 uM Template DNA X uL X pL DMSO optional 0 6 uL 1 5 uL 3 Phusion Hot Start II DNA Polymerase 0 2 uL 0 5 uL 0 02 U uL 2 U uL Optionally 5X Phusion Green GC Buffer can be used See section 4 2 for details The recommendation for final primer concentration is 0 5 uM but it can be varied in a range of 0 2 1 0 uM if needed Addition of DMSO is recommended for GC rich amplicons DMSO is not recommended for amplicons with very low GC or amplicons that are 220 kb Table 2 Cycling instructions 2 step protocol 3 step protocol Cycle step Cycles Temp Time Temp Time Initial 98ec 30s osc 30s 1 denaturation Denaturation 98 C 5 10s 98 C 5 10s Annealing X C 10 30 s see 5 3 25 35 Extension 72 C 15 30 s kb 72 C 15 30 s kb see 5 4 72 C 5 10 min 72 C 5 10 min 4 C hold 4 C hold Final Extension 4 Notes about reaction components 4 1 Enzyme The optimal amount of enzyme depends on the amount of template and the length of the PCR product Usually 1 unit of Phusion Hot Start II DNA Polymerase per 50 uL reaction volume gives good results but the optimal amount can range from 0 5 to 2 units per 50 uL reaction depending on the amplicon length and difficulty Do not exceed 2 U 50 pL 0 04 U L especially for amplicons that are gt 5 kb When cloning fragments amplified with Phusion Hot Start II DNA Polymera
5. ld e Repeat the PCR and Decrease annealing make sure that there are temperature no pipetting errors e Optimize enzyme e Use Thermo Scientific s concentration Tm calculator www thermoscientific com pcrwebtools Use fresh high quality Titrate DMSO 2 8 in the reaction see section 4 5 Denaturation temperature may be too low Optimal dNTPs denaturation temperature e Do not use dNTP mix or for most templates is 98 C primers that contain dUTP or higher or dITP Sample concentration may be too low Use more Denaturation time may be too long or too short Optimize denaturation time template e Check the purity and e Template DNA may be concentration of the damaged Use carefully primers purified template e Check primer design e Increase extension time Try using the alternative GC e Increase the number of cycles Buffer see section 4 2 Non specific products High molecular weight smears e Decrease enzyme e Vary denaturation concentration temperature see section see section 4 1 5 2 e Decrease extension time e Optimize Mg concentration see section 5 4 see section 4 3 e Reduce the total number e Reduce primer of cycles concentration e Increase annealing temperature or try 2 step protocol see section 5 3 Non specific products Low molecular weight discrete bands e Increase annealing e Optimize Mg concentration temperature see section 4 3 se
6. le strand and prevents complete denaturation of DNA Excess Mg can also stabilize spurious annealing of primers to incorrect template sites and decrease specificity Conversely inadequate Mg may lead to lower product yield The optimal Mg concentration also depends on the dNTP concentration the specific template DNA and the sample buffer composition In general the optimal Mg concentration is 0 5 to 1 mM over the total dNTP concentration for standard PCR If the primers and or template contain chelators such as EDTA or EGTA the apparent Mg optimum may be shifted to higher concentrations If further optimization is needed increase Mg2 concentration in 0 2 mM steps High quality dNTPs should be used for optimal performance with Phusion Hot Start Il DNA Polymerase The polymerase cannot read dUTP derivatives or dITP in the template strand so the use of these analogues or primers containing them is not recommended Due to the high processivity of Phusion Hot Start II DNA Polymerase there is no advantage of increasing dNTP concentrations For optimal results always use 200 uM of each dNTP 4 4 Template General guidelines for low complexity DNA e g plasmid lambda or BAC DNA are 1 pg 10 ng per 50 uL reaction volume For high complexity genomic DNA the amount of DNA template should be 50 250 ng per 50 uL reaction volume If cDNA synthesis reaction mixture is used as a source of template the volume of the template should not e
7. nditions 7 4 50 mM MgCl Solution F 510MG Both Phusion Buffers supply 1 5 mM MgCl at final reaction conditions If higher MgClz concentrations are desired use a 50 mM MgCl solution to increase the MgCl titer Using the following equation you can calculate the volume of 50 mM MgClz needed to attain the final MgClz concentration desired mM Mg 1 5 mM uL to add to a 50 uL reaction For example to increase the MgClz concentration to 2 0 mM add 0 5 mL of the 50 mM MgCl solution Because the PCR reactions can be quite sensitive to changes in the MgCle concentration it is recommended that the 50 mM MgCle stock solution is diluted 1 5 to 10 mM to minimize pipetting errors 7 5 Dimethyl sulfoxide DMSO 100 F 515 Note The freezing point of DMSO is 18 19 C so it does not melt on ice 8 References 1 Frey M amp Suppmann B 1995 Biochemica 2 34 35 2 Nord K et al 1997 Nature Biotechnol 15 772 777 3 Wikman M et al 2004 Protein Eng Des Sel 17 455 462 4 Chester N amp Marshak D R 1993 Anal Biochem 209 284 290 CERTIFICATE OF ANALYSIS Endonuclease contamination assay No endonuclease activity was observed after incubation of DNA polymerase with supercoiled plasmid DNA DNA amplification assay Performance in PCR is tested by the amplification of 2 3 and 7 5 kb genomic DNA and a 20 kb lambda DNA Quality authorized by Jurgita Zilinskiene TECHNICAL SUPPORT EMEA ts molbio eu th
8. optimal annealing temperature for Phusion Hot Start II DNA Polymerase may differ significantly from that of Taq based polymerases Always use the Tm calculator and instructions on website www thermoscientific com pcrwebtools to determine the Tm values of primers and optimal annealing temperature As a basic rule for primers gt 20 nt anneal for 10 30 seconds at a Tm 3 C of the lower Tm primer For primers lt 20 nt use an annealing temperature equal to the Tm of the lower Tm primer If necessary use a temperature gradient to find the optimal annealing temperature for each template primer pair combination The annealing gradient should extend up to the extension temperature two step PCR A 2 step protocol is recommended when primer Tm values are at least 69 C gt 20 nt or 72 C S20 nt when calculated with Thermo Scientifics Tm calculator In the 2 step protocol the combined annealing extension step should be performed at 72 C even when the primer Tm is gt 72 C 5 4 Extension The extension should be performed at 72 C The extension time depends on the length and complexity of the amplicon For low complexity DNA e g plasmid lambda or BAC DNA use an extension time of 15 seconds per 1 kb For high complexity genomic DNA 30 seconds per 1 kb is recommended For some cDNA templates the extension time can be increased up to 40 seconds per 1 kb to obtain optimal results 6 Troubleshooting No product at all or low yie
9. refully e Use 200 uM of each dNTP Do not use dUTP see 4 3 e Phusion DNA Polymerases produce blunt end DNA products 3 Guidelines for using Phusion Hot Start Il DNA Polymerase Carefully mix and centrifuge all tubes before opening to ensure homogeneity and improve recovery When using Phusion Hot Start II DNA Polymerase it is not necessary to perform the PCR setup on ice Prepare a master mix for the appropriate number of samples to be amplified The DNA polymerase should be pipetted carefully and gently as the high glycerol content 50 in the storage buffer may otherwise lead to pipetting errors Protocols optimized for Phusion DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions Due to the novel nature of Phusion Hot Start II DNA Polymerase the optimal reaction conditions may differ from PCR protocols for standard DNA polymerases Due to the high salt concentration in the reaction buffer Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures Please pay special attention to the conditions listed below when running your reactions Following the guidelines will ensure optimal enzyme performance Table 1 Pipetting instructions add items in this order Component 20 uL rxn 50 uL rxn Final conc H20 Add to 20 uL add to 50 uL a rnuson Green HF 4 uL 10 uL 1X 10 mM dNTPs 0 4 pL 1 uL ne Primer A X uL X uL 0 5 uM Pr
10. reversibly bound specific Affibody protein2 which inhibits the DNA polymerase activity at ambient temperatures thus preventing the amplification of non specific products In addition the Affibody ligand inhibits the 3 5 exonuclease activity of the polymerase preventing degradation of primers and template DNA during reaction setup At polymerization temperatures the Affibody molecule is released rendering the polymerase fully active Phusion Hot Start II DNA Polymerase does not require any separate activation step in the PCR protocol The 5X Phusion Green HF Buffer and 5X Phusion Green GC Buffer include a density reagent and two tracking dyes for direct loading of PCR products on a gel The colored buffer does not interfere with PCR performance and is compatible with downstream applications such as DNA sequencing ligation and restriction digestion For applications that require PCR product analysis by absorbance or fluorescence excitation we recommend using the colorless 5X Phusion HF Buffer F 518 or 5X Phusion GC Buffer F 519 or purifying the PCR product prior to analysis 2 Important Notes e Use Phusion DNA Polymerase at 0 5 1 0 U per 50 uL reaction volume Do not exceed 2 U 50 uL see 4 1 e Use 15 30 s kb for extension Do not exceed 1 min kb see 5 4 e Use 98 C for denaturation see 5 1 amp 5 2 e The annealing rules are different from many common DNA polymerases such as Tag DNA polymerases Read Section 5 3 ca
11. se blunt end cloning is recommended If TA cloning is required it can be performed by adding A overhangs to the blunt PCR product with Taq DNA Polymerase for example However before adding the overhangs it is very important to remove all Phusion Hot Start II DNA Polymerase by purifying the PCR product carefully Any remaining Phusion Hot Start II DNA Polymerase will degrade the A overhangs creating blunt ends again A detailed protocol for TA cloning of PCR fragments amplified with any of the Phusion DNA Polymerases can be found on website www thermoscientific com pcrcloning 4 2 Buffers Two buffers are provided with the enzyme 5X Phusion Green HF Buffer and 5X Phusion Green GC Buffer The error rate of Phusion Hot Start II DNA Polymerase in HF Buffer 4 4 x 107 is lower than that in GC Buffer 9 5 x 107 Therefore HF Buffer should be used as the default buffer for high fidelity amplification However GC Buffer can improve the performance of Phusion Hot Start II DNA Polymerase on some difficult or long templates such as GC rich templates or those with complex secondary structures For applications such as microarray or DHPLC where the DNA templates need to be free of detergents non colored detergent free reaction buffers are available for Phusion DNA Polymerases 4 3 Mg and dNTP The concentration of Mg is critical since Phusion Hot Start II DNA Polymerase is a magnesium dependent enzyme Excessive Mg stabilizes the DNA doub
12. xceed 10 of the final PCR reaction volume 4 5 PCR additives The recommended reaction conditions for GC rich templates include 3 DMSO as a PCR additive which aids in the denaturing of templates with high GC content For further optimization DMSO should be increased in 2 steps In some cases DMSO may also be required for supercoiled plasmids to relax for denaturation Other PCR additives such as formamide up to 3 glycerol and betaine are also compatible with Phusion Hot Start Il DNA Polymerase If high DMSO concentration is used the annealing temperature must be decreased as DMSO affects the melting point of the primers It has been reported that 10 DMSO decreases the annealing temperature by 5 5 6 0 C4 5 Notes about cycling conditions 5 1 Initial denaturation Denaturation should be performed at 98 C Due to the high thermostability of Phusion Hot Start II DNA Polymerase even higher than 98 C denaturation temperatures can be used We recommend a 30 second initial denaturation at 98 C for most templates Some templates may require longer initial denaturation time and the length of the initial denaturation time can be extended up to 3 minutes 5 2 Denaturation Keep the denaturation time as short as possible Usually 5 10 seconds at 98 C is enough for most templates Note the denaturation time and temperature may vary depending on the ramp rate and temperature control mode of the cycler 5 3 Primer annealing The
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