Team:LMU-Munich/Notebook/Protocols/20 PCR with Phusion Hot Start

PCR with Phusion Polymerase

Source: Finnzymes/Thermo scientific http://www.finnzymes.fi/pdf/phusion_hs2_datasheet_f549sl_1_2_low.pdf

Protocols optimized for Phusion® DNA Polymerase can be applied to Phusion Hot Start II DNA Polymerase reactions.

Table 1. Pipetting instructions (add items in this order).

(* Optionally 5x Phusion GC Buffer can be used. See section 4.2. for details.

(** The recommendation for final primer concentration is 0.5 μM, but it can be varied in a range of 0.2–1.0 μM 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 >20 kb.

Use Phusion DNA Polymerase at 0.5–1.0 U per 50 μl reaction volume. Do not exceed 2 U/50 μl. (See 4.1) Use 15–30 s/kb for extension. Do not exceed 1 min/kb. (See 5.4) Use 98°C for denaturation. (See 5.1 & 5.2) The annealing rules are different from many common DNA polymerases (such as Taq DNA polymerases). Read Section 5.3 carefully. Use 200 μM of each dNTP. Do not use dUTP.

4.4 Template General guidelines for low complexity DNA (e.g. plasmid, lambda or BAC DNA) are: 1 pg–10 ng per 50 μl reaction volume. For high complexity genomic DNA, the amount of DNA template should be 50–250 ng per 50 μl reaction volume. If cDNA synthesis reaction mixture is used as a source of template, the volume of the template should not exceed 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 II 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 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 Finnzymes’ website (www.finnzymes.com) to determine the Tm values of primers and optimal annealing temperature. As a basic rule, for primers >20 nt, anneal for 10–30 seconds at a Tm +3°C of the lower Tm primer. For primers ≤ 20 nt, Cycle step 2-step protocol 3-step protocol Temp. Time Temp. Time Cycles Initial denaturation 98°C 30 s 98°C 30 s 1 Denaturation Annealing (see 5.3) Extension (see 5.4) 98°C – 72°C 5–10 s – 15–30 s/kb 98°C X°C 72°C 5–10 s 10–30 s 15–30 s/kb 25–35 Final extension 72°C 4°C 5–10 min hold 72°C 4°C 5–10 min hold 1 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 (> 20 nt) or 72°C (≤ 20 nt) when calculated with Finnzymes’ Tm calculator. In the 2-step protocol the combined annealing/ extension step should be performed at 72°C even when the primer Tm is > 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 7. Component specifications 7.1 Phusion® Hot Start II High-Fidelity DNA Polymerase (F-549) Thermostable Phusion DNA Polymerase is isolated and purified from an E. coli strain 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 Affibodyencoding gene. Phusion Hot Start II DNA Polymerase is free of contaminating endo- and exonucleases. Storage buffer: 20 mM Tris-HCl (pH 7.4 at 25°C), 0.1 mM EDTA, 1 mM DTT, 100 mM KCl, stabilizers, 200 μg/ml BSA and 50 % glycerol. Unit definition: One unit is defined as the amount of enzyme that will incorporate 10 nmoles of dNTPs into acid-insoluble form at 74°C in 30 minutes under the stated assay conditions. Unit assay conditions: Incubation buffer: 25 mM TAPSHCl, pH 9.3 (at 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM β-mercaptoethanol, 100 μM dCTP, 200 μM each dATP, dGTP, dTTP. Incubation procedure: 20 μg activated calf thymus DNA and 0.5 μCi [α-32P] dCTP are incubated with 0.1 units of DNA polymerase in 50 μl incubation buffer at 74°C for 10 minutes. The amount of incorporated dNTPs is determined by trichloroacetic acid precipitation. DNA amplification test: Performance in PCR is tested by the amplification of 2.3 and 7.5 kb genomic DNA and 20 kb λ DNA. Exonuclease contamination assay: Incubation of 10 U for 4 hours at 72°C in 50 μl assay buffer with 1 μg sonicated [3H] DNA (2 x 105 cpm/μg) released < 1 % of radioactivity. Endonuclease contamination assay: No endonuclease activity was observed after incubation of 10 U of DNA polymerase with 1 μg of λ DNA in assay buffer at 72°C for 4 hours. 7.2 5x Phusion® HF Buffer (F-518) The 5x Phusion HF Buffer contains 7.5 mM MgCl2, which provides 1.5 mM MgCl2 in final reaction conditions. 7.3 5x Phusion® GC Buffer (F-519) The 5x Phusion GC Buffer contains 7.5 mM MgCl2, which provides 1.5 mM MgCl2 in final reaction conditions. Caution: Repeated freezing and thawing of the buffer can result in the precipitation or accumulation of MgCl2 in insoluble form. For consistent results, heat the buffer to 90°C for 10 min and vortex prior to use if needed, or store refrigerated. 7.4 50 mM MgCl2 Solution (F-510MG) Both Phusion Buffers supply 1.5 mM MgCl2 at final reaction conditions. If higher MgCl2 concentrations are desired, use a 50 mM MgCl2 solution to increase the MgCl2 titer. Using the following equation, you can calculate the volume of 50 mM MgCl2 needed to attain the final MgCl2 concentration: [desired mM Mg] – [1.5 mM] = μl to add to a 50 μl reaction. For example to increase the MgCl2 concentration to 2.0 mM, add 0.5 μl of the 50 mM MgCl2 solution. Because the No product at all or low yield • Repeat the PCR and make sure that there are no pipetting errors. • Use Finnzymes’ Tm calculator (www.finnzymes.com). • Use fresh high-quality dNTPs. • Do not use dNTP mix or primers that contain dUTP or dITP. • Sample concentration may be too low. Use more template. • Template DNA may be damaged. Use carefully purified template. • Increase extension time. • Increase the number of cycles. • Decrease annealing temperature. • Optimize enzyme concentration. • Titrate DMSO (2–8 %) in the reaction (see section 4.5). • Denaturation temperature may be too low. Optimal denaturation temperature for most templates is 98°C or higher. • Denaturation time may be too long or too short. Optimize denaturation time. • Check the purity and concentration of the primers. • Check primer design. • Try using the alternative GC Buffer (see section 4.2). Non-specific products - High molecular weight smears • Decrease enzyme concentration (see section 4.1). • Decrease extension time (see section 5.4). • Reduce the total number of cycles. • Increase annealing temperature or try 2-step protocol (see section 5.3). • Vary denaturation temperature (see section 5.2). • Optimize Mg2+ concentration (see section 4.3). • Reduce primer concentration. Non-specific products - Low molecular weight discrete bands • Increase annealing temperature (see section 5.3). • Shorten extension time (see section 5.4). • Reduce enzyme concentration (see section 4.1). • Optimize Mg2+ concentration (see section 4.3). • Titrate template amount. • Decrease primer concentration. • Design new primers. PCR reactions can be quite sensitive to changes in the MgCl2 concentration, it is recommended that the 50 mM MgCl2 stock solution is diluted 1:5 (to 10 mM) to minimize pipetting errors. 8. References 1. Frey M. & 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. & Marshak D.R. (1993) Anal. Biochem. 209: 284–290. Shipping and storage Phusion Hot Start II DNA Polymerase is shipped on gel ice. Upon arrival, store the components at −20°C. Phusion Hot Start II DNA Polymerase is stable for one year from the assay date when stored and handled properly. Warranty Finnzymes Oy warrants that its products will meet the specifications stated on the technical data section of the data sheets, and Finnzymes Oy agrees to replace the products free of charge if the products do not conform to the specifications. Notice for replacement must be given within 60 days of receipt. In consideration of the above commitments by Finnzymes Oy, the buyer agrees to and accepts the following conditions: • That this warranty is in lieu of all other warranties, express or implied; • That ALL WARRANTIES OF MERCHANTABILITY OR OF FITNESS FOR A PARTICULAR PURPOSE ARE HEREBY EXCLUDED AND WAIVED; • That the buyer’s sole remedy shall be to obtain replacement of the product free of charge from Finnzymes Oy; and • That this remedy is in lieu of all other remedies or claims for damages, consequential or otherwise, which the buyer may have against Finnzymes Oy. 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Limited license 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. The quality system of Finnzymes Oy is certified according to standard SFS-EN ISO9001:2008. Phusion® and DyNAzyme™ are trademarks or registered trademarks of Finnzymes Oy, a Thermo Fisher Scientific company. Affibody® is a registered trademark of Affibody AB, Sweden. Version 1.2, April 2010