Thermo Fisher Scientific Platinum SuperFi PCR Master Mix User guide

Brand
Thermo Fisher Scientific
Model
Platinum SuperFi PCR Master Mix
Type
User guide
Enzyme characteristics
Hot-start: Antibody
Length: Up to 20 kb
Fidelity vs. Taq:>100X
Format: Master Mix
PCR setup
Component 25-µL rxn 50-µL rxn Custom Final conc.
Water, nuclease-free to 25µL to 50µL to µL —
2X Platinum™ SuperFi™
PCR Master Mix112.5µL 25µL µL 1X
10µM forward primer 1.25µL 2.5µL µL 0.5µM
10µM reverse primer 1.25µL 2.5µL µL 0.5µM
Template DNA2varies varies varies
5X SuperFi™ GC Enhancer
(optional)35µL 10µL µL 1X
1 Provides MgCl2 at a nal concentration of 1.5 mM in the reaction.
2 5–50 ng gDNA or 1 pg–10 ng plasmid DNA (see Optimization strategies for more
information).
3 Recommended for targets with >65% GC sequences.
PCR protocol
See page 2 and page 3 to prepare and run your PCR experiment.
Optimization strategies and troubleshooting
Click here for guidelines to optimize your PCR experiment.
Click here for guidelines to troubleshoot your PCR experiment.
Purchaser notification
Click here for Limited warranty, Disclaimer, and Licensing information.
Package
contents
Catalog number
12358-010
12358-050
12358-250
Size
100 rxns
500 rxns
5 × 500 rxns
Kit contents
Storage
conditions ∤Store all contents at –20°C.
Required
materials
∤Template: gDNA, plasmid DNA, phage DNA, cDNA
∤Forward and reverse gene-specic primers
∤Invitrogen™ E-Gel™ General Purpose Gels, 1.2%
(Cat.No.G5018-01)
∤Invitrogen™ TrackIt™ 1 kb Plus DNA Ladder
(Cat.No.10488-085)
∤0.2 or 0.5-mL nuclease-free microcentrifuge tubes
∤Gel loading buffer
Timing Varies depending on amplicon length
Selection
guide
PCR Enzymes and Master Mixes
Go online to view related products.
Product
description
∤Invitrogen™ Platinum™ SuperFi™ PCR Master Mix is a ready-
to-use mixture of DNA polymerase, salts, magnesium, and
dNTPs for efcient PCR amplication. It retains all the
features of the Platinum™ SuperFi™ DNA Polymerase.
∤ Platinum™ SuperFi™ DNA Polymerase is a proofreading DNA
polymerase that combines delity with Platinum™ hot-start
technology, and is ideally suited for cloning, mutagenesis,
and other applications.
∤Platinum™ hot-start technology inhibits DNA polymerase
activity at ambient temperatures, allowing room temperature
reaction setup and storage of pre-assembled PCR reactions
for up to 24 hours prior to the PCR. Enzyme activity is
restored after the initial denaturation step.
∤Platinum™ SuperFi™ DNA Polymerase has both 5' to 3'
polymerase and 3' to 5' exonuclease activities, but lacks 5' to 3'
exonuclease activity. It produces blunt end DNA products.
∤Platinum™ SuperFi™ PCR Master Mix is supplied with a
separate vial of SuperFi™ GC Enhancer designed for GC-rich
templates (>65% GC).
Important
guidelines Click here for important PCR guidelines.
Online
resources
Visit our product page for additional information and protocols.
For support, visit thermosher.com/support.
For Research Use Only. Not for use in diagnostic procedures.
USER GUIDE Pub. No. MAN0014883 Rev. B
Invitrogen™ Platinum™ SuperFi™ PCR Master Mix
Print Options
-2-
The example PCR procedure below shows appropriate volumes for a single 50-μL reaction. For multiple reactions, prepare a master mix of components common to all reactions to
minimize pipetting error, then dispense appropriate volumes into each 0.2–0.5-mL PCR tube prior to adding template DNA and primers.
Steps Action Procedure details
1 Thaw reagents Thaw, mix, and briey centrifuge each component before use. Avoid generating bubbles when mixing the
Master Mix.
2 Prepare PCR master mix
Add the following components to each PCR tube.
Note: Consider the volumes for all components listed in steps 2 and 3 to determine the correct amount of
water required to reach your nal reaction volume.
Component 50-µL rxn Final conc.
Water, nuclease-free to 50µL
2X Platinum™ SuperFi™ PCR Master Mix 25µL 1X
5X SuperFi™ GC Enhancer (optional)110µL 1X
1 Recommended for targets with >65% GC sequences.
Mix and then briey centrifuge the components.
3 Add template DNA and primers
Add your template DNA and primers to each tube for a nal reaction volume of 50L.
Component 50-µL rxn Final conc.
10µM forward primer 2.5µL 0.5µM
10µM reverse primer 2.5µL 0.5µM
Template DNA1varies varies
1 Optimal amount of low complexity DNA (plasmid, phage, BAC DNA) is 1 pg–10 ng per 50µL reaction, but it can be
varied from 0.1 pg to 50 ng per 50µL reaction. Optimal amount of genomic DNA is 5–50 ng per 50µL reaction, but it can
be varied from 0.1 ng to 250 ng per 50µL reaction.
Cap each tube, mix, and then briey centrifuge the contents.
1 June 2017
For support, visit thermofisher.com/support.
-3-
Steps Action Procedure details
4
Incubate reactions in a
thermal cycler
Step
2-step protocol (<10kb) 3-step protocol (<10kb) Long PCR (>10kb)
Temp. Time Temp. Time Temp. Time
Initial denaturation 98°C 30 sec 98°C 30 sec 95°C 2 min
25–35
PCR
cycles
Denature 98°C 5–10 sec 98°C 5–10 sec 95°C 10 sec
Anneal1— — varies 10 sec varies 10 sec
Extend 72°C 15–30 sec/kb 72°C 15–30 sec/kb 68°C 30 sec/kb
Final extension 72°C 5 min 72°C 5 min 68°C 5 min
4°C hold 4°C hold 4°C hold
1 IMPORTANT! Always use Tm calculator on our website at www.thermosher.com/tmcalculator to calculate Tm of your
primers and the recommended annealing temperature.
Note: Refer to “Optimization strategies”, page 1, for guidelines to optimize cycling conditions.
5
Add gel loading buffer and
analyze with gel
electrophoresis
Add gel loading buffer to 10µL of PCR product, mix, and briey centrifuge the contents.
Analyze the sample using agarose gel electrophoresis.
Use your PCR product immediately in down-stream applications, or store it at –20°C.
Enzyme characteristics
Hot-start: Antibody
Length: Up to 20 kb
Fidelity vs. Taq:>100X
Format: Master Mix
PCR setup
Component 25-µL rxn 50-µL rxn Custom Final conc.
Water, nuclease-free to 25µL to 50µL to µL —
2X Platinum™ SuperFi™
PCR Master Mix112.5µL 25µL µL 1X
10µM forward primer 1.25µL 2.5µL µL 0.5µM
10µM reverse primer 1.25µL 2.5µL µL 0.5µM
Template DNA2varies varies varies
5X SuperFi™ GC Enhancer
(optional)35µL 10µL µL 1X
1 Provides MgCl2 at a nal concentration of 1.5 mM in the reaction.
2 5–50 ng gDNA or 1 pg–10 ng plasmid DNA (see Optimization strategies for more
information).
3 Recommended for targets with >65% GC sequences.
PCR protocol
See page 2 and page 3 to prepare and run your PCR experiment.
Optimization strategies and troubleshooting
Click here for guidelines to optimize your PCR experiment.
Click here for guidelines to troubleshoot your PCR experiment.
Purchaser notification
Click here for Limited warranty, Disclaimer, and Licensing information.
Package
contents
Catalog number
12358-010
12358-050
12358-250
Size
100 rxns
500 rxns
5 × 500 rxns
Kit contents
Storage
conditions ∤Store all contents at –20°C.
Required
materials
∤Template: gDNA, plasmid DNA, phage DNA, cDNA
∤Forward and reverse gene-specic primers
∤Invitrogen™ E-Gel™ General Purpose Gels, 1.2%
(Cat.No.G5018-01)
∤Invitrogen™ TrackIt™ 1 kb Plus DNA Ladder
(Cat.No.10488-085)
∤0.2 or 0.5-mL nuclease-free microcentrifuge tubes
∤Gel loading buffer
Timing Varies depending on amplicon length
Selection
guide
PCR Enzymes and Master Mixes
Go online to view related products.
Product
description
∤Invitrogen™ Platinum™ SuperFi™ PCR Master Mix is a ready-
to-use mixture of DNA polymerase, salts, magnesium, and
dNTPs for efcient PCR amplication. It retains all the
features of the Platinum™ SuperFi™ DNA Polymerase.
∤ Platinum™ SuperFi™ DNA Polymerase is a proofreading DNA
polymerase that combines delity with Platinum™ hot-start
technology, and is ideally suited for cloning, mutagenesis,
and other applications.
∤Platinum™ hot-start technology inhibits DNA polymerase
activity at ambient temperatures, allowing room temperature
reaction setup and storage of pre-assembled PCR reactions
for up to 24 hours prior to the PCR. Enzyme activity is
restored after the initial denaturation step.
∤Platinum™ SuperFi™ DNA Polymerase has both 5' to 3'
polymerase and 3' to 5' exonuclease activities, but lacks 5' to 3'
exonuclease activity. It produces blunt end DNA products.
∤Platinum™ SuperFi™ PCR Master Mix is supplied with a
separate vial of SuperFi™ GC Enhancer designed for GC-rich
templates (>65% GC).
Important
guidelines Click here for important PCR guidelines.
Online
resources
Visit our product page for additional information and protocols.
For support, visit thermosher.com/support.
For Research Use Only. Not for use in diagnostic procedures.
USER GUIDE Pub. No. MAN0014883 Rev. B
Invitrogen™ Platinum™ SuperFi™ PCR Master Mix
n
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Final conc.
to µ
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Click here for Limited warranty, Disclaimer, and Licensing information.
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Important
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Important guidelines
∤The annealing rules for Invitrogen™ Platinum™ SuperFi™ DNA Polymerase are different from many common DNA polymerases (such as
Taq DNA polymerases). For optimal results, use the Tm calculator on our website at www.thermosher.com/tmcalculator.
∤Carefully mix and centrifuge all tubes before opening to ensure homogeneity and to improve recovery. Prepare a master mix for the
appropriate number of samples to be amplied.
∤When using Platinum™ SuperFi™ PCR Master Mix, it is not necessary to perform the PCR set up on ice.
∤Pipet the Platinum™ SuperFi™ PCR Master Mix carefully and gently. Otherwise, the high glycerol content (50%) in the storage buffer
may lead to pipetting errors.
∤Platinum™ SuperFi™ DNA Polymerase produces blunt end DNA products.
∤The polymerase cannot read dUTP-derivatives or dITP in the template strand. Therefore, we do not recommend the use of these
analogues or primers that contain them.
∤Take precautions to avoid cross-contamination by using aerosol-resistant barrier tips and by analyzing PCR products in a separate area
from PCR assembly.
∤Use 15–30 seconds/kb for extension. Do not exceed 1minute/kb.
∤Use the SuperFi™ GC Enhancer to improve amplication of DNA targets containing GC-rich sequences (>65% GC).
∤For a streamlined protocol, we recommend using Invitrogen™ Platinum™ SuperFi™ Green PCR Master Mix (Cat.No. 12359-010), which
contains tracking dyes and a density reagent for direct loading of PCR products on gels.
CLOSE
>>
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me character
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cs
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Kit contents
Reagents provided are sufcient for 100, 500, or 2500 amplication reactions of 50 L each.
Kit sizes
Component 100 reactions 500 reactions 2500 reactions
2X Platinum™ SuperFi™ PCR Master Mix 2 × 1.25mL 10 × 1.25mL 50 × 1.25mL
5X SuperFi™ GC Enhancer 1.25mL 4 × 1.25mL 20 × 1.25mL
Water, nuclease-free 2 × 1.25mL 10 × 1.25mL 50 × 1.25mL
CLOSE
>>
-2-
The example PCR procedure below shows appropriate volumes for a single 50-μL reaction. For multiple reactions, prepare a master mix of components common to all reactions to
minimize pipetting error, then dispense appropriate volumes into each 0.2–0.5-mL PCR tube prior to adding template DNA and primers.
Steps Action Procedure details
1 Thaw reagents Thaw, mix, and briey centrifuge each component before use. Avoid generating bubbles when mixing the
Master Mix.
2 Prepare PCR master mix
Add the following components to each PCR tube.
Note: Consider the volumes for all components listed in steps 2 and 3 to determine the correct amount of
water required to reach your nal reaction volume.
Component 50-µL rxn Final conc.
Water, nuclease-free to 50µL
2X Platinum™ SuperFi™ PCR Master Mix 25µL 1X
5X SuperFi™ GC Enhancer (optional)110µL 1X
1 Recommended for targets with >65% GC sequences.
Mix and then briey centrifuge the components.
3 Add template DNA and primers
Add your template DNA and primers to each tube for a nal reaction volume of 50L.
Component 50-µL rxn Final conc.
10µM forward primer 2.5µL 0.5µM
10µM reverse primer 2.5µL 0.5µM
Template DNA1varies varies
1 Optimal amount of low complexity DNA (plasmid, phage, BAC DNA) is 1 pg–10 ng per 50µL reaction, but it can be
varied from 0.1 pg to 50 ng per 50µL reaction. Optimal amount of genomic DNA is 5–50 ng per 50µL reaction, but it can
be varied from 0.1 ng to 250 ng per 50µL reaction.
Cap each tube, mix, and then briey centrifuge the contents.
-2
-
The e
x
o
ns to
mini
m
1
2
3
a
n
x
ample PCR procedure below shows appropriate volumes for a sin
g
le
5
0-μ
L
reaction. For multi
p
le reactions,
p
re
pa
re a master mix of com
po
nents common to all reacti
o
m
ize pipettin
g
error, then dispense appropriate volumes into each 0.2–0.5-mL PCR tube prior to addin
g
template DNA and primers
.
S
S
S
S
S
S
S
S
t
t
t
t
t
t
e
e
e
e
ep
s
A
c
ti
on
P
r
ocedu
r
e
de
t
a
il
s
Th
aw reagents Thaw, mix, and briey centrifuge each component before use. Avoid generating bubbles when mixing the
M
a
s
t
e
r Mix.
Prepare PCR master mix
Add the following components to each PCR tube
.
N
ote: Consider the volumes for all com
p
onents listed in ste
p
s 2 and 3 to determine the correct amount o
f
water required to reach your nal reaction volume
.
Component 50-µL rx
n
Fin
a
l
co
n
c
.
W
ater, nuclease-
f
ree to 50µ
L
2
X Platinu
m
™
S
up
erF
i
™
P
C
R Master Mix
2
5µ
L
1
X
5X
S
u
p
er
Fi
™
GC Enhancer
(
optional
)
1
1
0µL 1X
1
Recommended for targets with >65% GC sequence
s.
Mix and then brie
y centri
f
uge the components.
A
dd template DNA and pr
i
me
rs
Add your template DNA and primers to each tube
f
or a
nal reaction volume o
f
50L.
Com
p
onent 50-
µ
L rxn
F
inal conc
.
10µM
f
orward primer 2.5
µL
0
.5
µM
10µM reverse
pr
imer 2.5
µL
0
.5
µM
Temp
l
ate D
NA
1
var
i
e
s
var
i
es
1
Optimal amount o
f
low complexity DNA (plasmid, phage, BAC DNA) is 1 pg–10 ng per 50µL reaction, but it can be
varied from 0.1 p
g
to 50 n
g
per 50µL reaction. Optimal amount of
g
enomic DNA is 5–50 n
g
per 50µL reaction, but it c
a
be varied from 0.1 ng to 250 ng per 50µL reaction.
C
ap each tube, mix, and then brie
y centri
f
uge the contents.
Optimization strategies
Notes about reaction components
Polymerase: Optimal amount of Platinum
™
SuperFi
™
DNA Polymerase is 1U per
50L reaction in most cases, but it can be varied in a range of 0.5–2.0U per 50L
reaction.
Do not exceed 2U of polymerase per 50L reaction (0.04U/µL).
Mg
2+
: SuperFi
™
Buffer provides MgCl
2
at a  nal concentration of 1.5mM in the
reaction.
∤
If the primers and/or the template contain chelators such as EDTA or EGTA, the
apparent Mg
2+
optimum may be shifted to higher concentrations.
∤
If further optimization is needed, increase the Mg
2+
concentration in 0.2mM
increments.
dNTPs: Use high quality dNTPs for optimal performance and highest  delity.
∤
The polymerase cannot read dUTP derivatives or dITP in the template strand.
Therefore, we do not recommend the use of these analogues or primers that
contain them.
∤
Due to high processivity of the enzyme, there is no advantage of increasing the
dNTP concentration. For optimal results, always use 200µM of each dNTP.
Primers: We recommend a  nal primer concentration of 0.5M, but this can be
varied in a range of 0.2–1.0M, if needed.
Template: Optimal amount of low complexity DNA (plasmid, phage, BAC DNA)
is 1pg–10ng per 50µL reaction, but it can be varied from 0.1pg to 50ng per 50µL
reaction. Optimal amount of genomic DNA is 5–50ng per 50µL reaction, but it can
b
e varied from 0.1ng to 250ng per 50µL reaction.
SuperFi
™
GC Enhancer: Use the SuperFi
™
GC Enhancer to improve ampli cation of
DNA targets containing problematic or GC-rich sequences (>65%GC).
Notes about cycling parameters
Make sure that the heated lid temperature is set several degrees above 98°C to
avoid sample condensation. The lid can be pre-heated before putting the samples in
the thermocycler.
Initial denaturation: 30-second initial denaturation at 98°C is suf cient for most
templates. Some templates may require longer initial denaturation time, which
can be extended up to 3minutes. For targets >10kb, we recommend an initial
denaturation of 2minutes at 95 °C.
Denaturation: Keep the denaturation time as short as possible. Usually
5–10seconds at 98°C is suf cient for most templates. For targets >10kb, reduce the
denaturation temperature to 95°C.
Annealing: Always use the T
m
calculator available on our website
(www.thermo sher.com/tmcalculator) to calculate the T
m
of your primers and the
recommended annealing temperature.
∤
If necessary, use a temperature gradient to further optimize and empirically
determine the ideal annealing temperature for each template-primer pair
combination. The annealing gradient should start with a temperature 6–10°C
lower than the annealing temperature generated by the calculator and increased
up to the extension temperature (two-step PCR).
∤
We recommend a 2-step protocol when the primer T
m
values are at least 69°C
(>20nt) or 72°C (≤20nt), when calculated with our T
m
calculator. In the 2-step
protocol, the combined annealing/extension step should be performed at 72°C
even when the primer T
m
is >72°C.
Extension: Extension time depends on amplicon length and complexity.
∤
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, use an extension time of 30seconds per 1kb.
∤
For some cDNA templates, the extension time can be increased up to 40seconds
per 1kb to obtain optimal results.
∤
Reduce the extension temperature to 68°C for targets >10kb.
CLOSE
>>
1 June 2017
For support, visit thermofisher.com/support.
-3-
Steps Action Procedure details
4
Incubate reactions in a
thermal cycler
Step
2-step protocol (<10kb) 3-step protocol (<10kb) Long PCR (>10kb)
Temp. Time Temp. Time Temp. Time
Initial denaturation 98°C 30 sec 98°C 30 sec 95°C 2 min
25–35
PCR
cycles
Denature 98°C 5–10 sec 98°C 5–10 sec 95°C 10 sec
Anneal1— — varies 10 sec varies 10 sec
Extend 72°C 15–30 sec/kb 72°C 15–30 sec/kb 68°C 30 sec/kb
Final extension 72°C 5 min 72°C 5 min 68°C 5 min
4°C hold 4°C hold 4°C hold
1 IMPORTANT! Always use Tm calculator on our website at www.thermosher.com/tmcalculator to calculate Tm of your
primers and the recommended annealing temperature.
Note: Refer to “Optimization strategies”, page 1, for guidelines to optimize cycling conditions.
5
Add gel loading buffer and
analyze with gel
electrophoresis
Add gel loading buffer to 10µL of PCR product, mix, and briey centrifuge the contents.
Analyze the sample using agarose gel electrophoresis.
Use your PCR product immediately in down-stream applications, or store it at –20°C.
1
Jun
For
Ste
ps
4
Incubat
t
h
e
r
Lon
g
PCR (>10kb)
T
emp. T
i
m
e
95°
C
2
min
9
5°
C
1
0 se
c
varies
1
0 se
c
6
8°
C
3
0 sec/k
b
6
8°
C
5
mi
n
4
°
C
h
o
ld
a
lculator to ca
l
cu
l
ate T
m
of your
n
g
conditions.
5
Add gel l
o
a
na
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For Research Use Only. Not for use in diagnostic procedures.
Limited product warranty
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Technologies’ website at www.thermosher.com/us/en/home/global/terms-and-conditions.html. If you have any questions, please contact Life Technologies at
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Troubleshooting
Observation Recommended action
No product at all or low
yield. ∤Make sure that there are no pipetting errors.
∤Do not use primers or template that contain dUTP or dITP.
∤Titrate template amount.
∤Template DNA may be damaged. Use carefully puried template.
∤Increase extension time.
∤Increase the number of cycles.
∤Decrease annealing temperature.
∤Optimize denaturation time and temperature.
∤Check the purity and concentration of the primers.
∤Check primer design.
∤Try using the SuperFi™ GC Enhancer
Non specic products–
High molecular weight
smears
∤Decrease extension time.
∤Reduce the total number of cycles.
∤Increase annealing temperature or try 2-step protocol.
∤Optimize denaturation time and temperature.
∤Reduce enzyme concentration.
∤Reduce primer concentration.
∤Check primer design
Non specic products–
Low molecular weight
smears
∤Increase annealing temperature.
∤Decrease enzyme concentration.
∤Decrease extension time.
∤Titrate template amount.
∤Decrease primer concentration.
∤Check primer design
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Thermo Fisher Scientific Platinum SuperFi PCR Master Mix User guide

Brand
Thermo Fisher Scientific
Model
Platinum SuperFi PCR Master Mix
Type
User guide
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