Team:LMU-Munich/Notebook/Protocols/20 PCR with Phusion Hot Start
From 2010.igem.org
(New page: {{:Team:LMU-Munich/Templates/Page Header}} ==PCR with Phusion Polymerase== Source: Finnzymes/Thermo scientific http://www.finnzymes.fi/pdf/phusion_hs2_datasheet_f549sl_1_2_low.pdf Proto...) |
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Use 200 μM of each dNTP. Do not use dUTP. | Use 200 μM of each dNTP. Do not use dUTP. | ||
- | 4.4 Template | + | <b>Cycling Program</b> |
+ | |||
+ | (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 | ||
+ | | | ||
+ | |- | ||
+ | |} | ||
+ | |||
+ | <b>4.4 Template</b> | ||
+ | |||
General guidelines for low complexity DNA (e.g. plasmid, | General guidelines for low complexity DNA (e.g. plasmid, | ||
lambda or BAC DNA) are: 1 pg–10 ng per 50 μl reaction | lambda or BAC DNA) are: 1 pg–10 ng per 50 μl reaction | ||
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of template, the volume of the template should not exceed | of template, the volume of the template should not exceed | ||
10 % of the final PCR reaction volume. | 10 % of the final PCR reaction volume. | ||
- | 4.5 PCR additives | + | |
+ | <b>4.5 PCR additives</b> | ||
+ | |||
The recommended reaction conditions for GC-rich templates | The recommended reaction conditions for GC-rich templates | ||
include 3 % DMSO as a PCR additive, which aids in the | include 3 % DMSO as a PCR additive, which aids in the | ||
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(up to 3 %), glycerol and betaine are also compatible with | (up to 3 %), glycerol and betaine are also compatible with | ||
Phusion Hot Start II DNA Polymerase. | Phusion Hot Start II DNA Polymerase. | ||
+ | |||
If high DMSO concentration is used, the annealing | If high DMSO concentration is used, the annealing | ||
temperature must be decreased, as DMSO affects the melting | temperature must be decreased, as DMSO affects the melting | ||
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decreases the annealing temperature by 5.5–6.0°C4. | decreases the annealing temperature by 5.5–6.0°C4. | ||
- | 5. Notes about cycling conditions | + | <b>5. Notes about cycling conditions</b> |
- | + | ||
- | Denaturation should be performed at 98°C. | + | Denaturation should be performed at 98°C. |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
- | + | ||
Keep the denaturation time as short as possible. Usually 5–10 | Keep the denaturation time as short as possible. Usually 5–10 | ||
- | seconds at 98°C is enough for most templates. | + | seconds at 98°C is enough for most templates. |
- | + | ||
- | + | ||
- | + | ||
The optimal annealing temperature for Phusion Hot Start II | The optimal annealing temperature for Phusion Hot Start II | ||
DNA Polymerase may differ significantly from that of Taq-based | DNA Polymerase may differ significantly from that of Taq-based | ||
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on Finnzymes’ website (www.finnzymes.com) to determine | on Finnzymes’ website (www.finnzymes.com) to determine | ||
the Tm values of primers and optimal annealing temperature. | the Tm values of primers and optimal annealing temperature. | ||
+ | |||
As a basic rule, for primers >20 nt, anneal for 10–30 seconds | As a basic rule, for primers >20 nt, anneal for 10–30 seconds | ||
- | at a Tm +3°C of the lower Tm primer. | + | at a Tm +3°C of the lower Tm primer. |
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{{:Team:LMU-Munich/Templates/Page Footer}} | {{:Team:LMU-Munich/Templates/Page Footer}} |
Latest revision as of 10:16, 21 September 2010
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.
Cycling Program
(3 step protocol)
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.
PCR with Phusion Polymerase
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
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