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

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==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).

{|
|Component 
|50 μl reaction
|20 μlreaction
|Final conc.
|-
|H2O 
|add to 50 μl 
|add to 20 μl
|
|-
|5x Phusion® HF Buffer* 
|10 μl 
|4 μl 
|1x
|-
|10 mM dNTPs 
|1 μl 
|0.4 μl 
|200 μM each
|-
|primer A** 
|x μl 
|x μl 
|0.5 μM
|-
|primer B** 
|x μl 
|x μl 
|0.5 μM
|-
|template DNA 
|x μl 
|x μl
|
|-
|(DMSO***, optional) 
|(1.5 μl) 
|(0.6 μl) 
|(3 %)
|-
|Phusion® Hot Start IIDNA Polymerase (2 U/μl)
|0.5 μl 
|0.2 μl 
|0.02 U/μl
|-
|}

(* 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.
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PURPOSE ARE HEREBY EXCLUDED AND WAIVED;
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Finnzymes Oy recommends that the buyer and other persons using the products follow the
Guidelines for Research involving Recombinant DNA Molecules (NIH guidelines) Federal
Register, July 5, 1994 (59 FR 34496) and any amendments thereto. Finnzymes Oy disclaims
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Version 1.2, April 2010
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