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.
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 (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.
|
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