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


PCR with Phusion Polymerase

Source: Finnzymes/Thermo scientific

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.

Cycling Program

(3 step protocol)

Cycle step Temp. Time Cycles
Initial denaturation 98°C 30 s 1
Denaturation 98°C 5–10 s
Annealing (see 5) X°C 10–30 s 25-30
Extension 72°C 15–30 s/kb
Final extension 72°C 5–10 min 1
Pause 4°C hold

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

Denaturation should be performed at 98°C.

Keep the denaturation time as short as possible. Usually 5–10 seconds at 98°C is enough for most templates.

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 ( 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.