Thermo Fisher Scientific Platinum SuperFi DNA Polymerase User guide

Brand
Thermo Fisher Scientific
Model
Platinum SuperFi DNA Polymerase
Type
User guide
USER GUIDE Pub. No. MAN0014882 Rev. B
Invitrogen™ Platinum™ SuperFi™ DNA Polymerase
Enzyme characteristics
Hot-start: Antibody
Length: Up to 20kb
Fidelity vs. Taq:>100X
Format: Separate components
PCR setup
Component 25-µL rxn 50-µL rxn Custom Final conc.
Water, nuclease-free to 25µL to 50µL to µL —
5X SuperFi™ Buffer15µL 10µL µL 1X
10mM dNTP mix 0.5µL 1 µL µL 0.2mM each
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
Platinum™ SuperFi™ DNA
Polymerase (2U/µL) 0.25µL 0.5µL µL 0.02U/L
1 Includes 7.5mM MgCl2.
2 5–50ng gDNA or 1pg–10ng plasmid DNA (see “Optimization strategies”, below, 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
12351-010
12351-050
12351-250
Size
100Units
500Units
5 × 500Units
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™ 10mM dNTP Mix (Cat.No.18427-088)
∤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
∤Water, nuclease-free
Timing Varies depending on amplicon length.
Selection
guide
PCR Enzymes and Master Mixes
Go online to view related products.
Product
description
∤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 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™ DNA Polymerase 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.
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 before adding template DNA and primers.
Steps Action Procedure details
1 Thaw reagents Thaw, mix, and briey centrifuge each component before use.
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
5X SuperFi™ Buffer110µL 1X
10mM dNTP mix 1 µL 0.2mM each
5X SuperFi™ GC Enhancer (optional)210µL 1X
Platinum™ SuperFi™ DNA Polymerase 0.5µL 0.02U/L
1 Includes 7.5mM MgCl2.
2 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 the Tm calculator on our website at www.thermosher.com/tmcalculator to calculate the 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.
USER GUIDE Pub. No. MAN0014882 Rev. B
Invitrogen™ Platinum™ SuperFi™ DNA Polymerase
Enzyme characteristics
Hot-start: Antibody
Length: Up to 20kb
Fidelity vs. Taq:>100X
Format: Separate components
PCR setup
Component 25-µL rxn 50-µL rxn Custom Final conc.
Water, nuclease-free to 25µL to 50µL to µL —
5X SuperFi™ Buffer15µL 10µL µL 1X
10mM dNTP mix 0.5µL 1 µL µL 0.2mM each
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
Platinum™ SuperFi™ DNA
Polymerase (2U/µL) 0.25µL 0.5µL µL 0.02U/L
1 Includes 7.5mM MgCl2.
2 5–50ng gDNA or 1pg–10ng plasmid DNA (see “Optimization strategies”, below, 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
12351-010
12351-050
12351-250
Size
100Units
500Units
5 × 500Units
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™ 10mM dNTP Mix (Cat.No.18427-088)
∤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
∤Water, nuclease-free
Timing Varies depending on amplicon length.
Selection
guide
PCR Enzymes and Master Mixes
Go online to view related products.
Product
description
∤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 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™ DNA Polymerase 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.
US
ER
GU
ID
E
Pub
.
No
.
M
AN
00
14
882
Re
v.
B
Inv
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o
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nz
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i
cs
H
ot-start
:
Antibod
y
n
C
usto
m
Final conc.
to µ
L
—
µL
1X
µL
0
.2mM
e
a
ch
µL
0.5
µM
Pu
r
c
ha
se
r n
o
ti
fic
ati
o
n
C
lick here for Limited Warrant
y
, Disclaimer, and Licensin
g
information
.
P
ac
k
a
ge
co
n
te
n
ts
Catalo
g
numbe
r
1
235
1
-
0
1
0
1
2
351
-
0
50
1
2
351
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iz
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100
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500 U it
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it
co
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S
torag
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d
iti
o
n
s
∤
Store a
ll
co
n
Requ
i
red
mat
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∤
Template: g
D
∤
F
orward an
d
∤
I
nvitroge
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™
∤
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nvitroge
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™
(Cat.No.G
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∤
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nvitroge
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(
Cat.No.10
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∤
0.2 or 0.5-m
L
∤
Gel loading
templates
(>65%
GC)
.
I
mportant
g
uidelines Click here
f
or important PCR guidelines
.
Online
resource
s
V
isit our pro
d
uct page for additional information and protocols.
F
or su
pp
ort, visit thermosher.com/su
pp
ort
.
For Research Use Only. Not for use in diagnostic procedures.
Cli
c
k
h
ere
f
or gu
id
e
li
nes to trou
bl
es
h
oot your
PCR
exper
i
ment.
Gel
loading
se
pa
rate vial of S
up
er
Fi
™
GC Enhancer des
ig
ned for GC-rich
templates (>65% GC
)
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.
∤Platinum™ SuperFi™ DNA Polymerase produces blunt end DNA products.
∤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™ DNA Polymerase, it is not necessary to perform the PCR set up on ice.
∤Pipet the Platinum™ SuperFi™ DNA Polymerase carefully and gently. Otherwise, the high glycerol content (50%) in the storage buffer may lead to pipetting errors.
∤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 DNA Polymerase (Cat. No. 12357-010) with 5XSuperFi™ Green Buffer,
which contains tracking dyes and a density reagent for direct loading of PCR products on gels.
CLOSE
>>
Len
g
t
h
:U
p
to 20k
b
Fidelity vs.
T
a
q
:
>1
00
X
F
ormat:
S
e
pa
rate com
po
nent
s
P
CR setup
C
om
p
onent 25-
µ
L rx
n
50-
µ
L rx
n
W
ate
r,
nuclease-free
t
o 25µ
L
to 50
µL
5X Su
p
erFi
™
B
uf
fe
r
1
5
µL
1
0
µ
L
10
mM d
N
TP mi
x
0.5
µL
1
µL
10µM forward
p
rimer
1
.25
µL
2
.5
µL
50
0
Un
it
s
5
×
500
U
nit
s
n
tents at –20°C.
D
NA, p
l
asmi
d
DNA, phage DNA, cDNA
d
reverse gene-specic primer
s
10mM dNTP Mix
(
Cat.No.18427-088
)
E-Ge
l
™
Genera
l
Pur
po
se Ge
l
s, 1.2%
5
018-01)
Tra
c
kI
t
™
1kb Plu
s
D
N
A Ladd
e
r
4
88
-
0
85
)
L
nuclease-
f
ree microcentri
f
uge tubes
bu
ff
er
buffer
Kit contents
Reagents provided are sufcient for 100, 500, or 2500 amplication reactions of 50 L each.
Kit sizes
Component 100 Units 500 Units 2500 Units
Platinum™ SuperFi™ DNA Polymerase
(2U/µL)
50 µL 250 µL 5 × 250 µL
5X SuperFi™ Buffer 2 × 1.25 mL 6 × 1.25 mL 30 × 1.25 mL
5X SuperFi™ GC Enhancer 1.25 mL 4 × 1.25 mL 20 × 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 before adding template DNA and primers.
Steps Action Procedure details
1 Thaw reagents Thaw, mix, and briey centrifuge each component before use.
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
5X SuperFi™ Buffer110µL 1X
10mM dNTP mix 1 µL 0.2mM each
5X SuperFi™ GC Enhancer (optional)210µL 1X
Platinum™ SuperFi™ DNA Polymerase 0.5µL 0.02U/L
1 Includes 7.5mM MgCl2.
2 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
-
2
-
The example PCR procedure below shows appropriate volumes for a sin
g
le
5
0-μ
L
reaction. For multi
p
le reactions,
p
re
p
are a master mix of com
p
onents common to all reactions to
minimize pipettin
g
error, then dispense appropriate volumes into each 0.2–0.5-mL PCR tube before addin
g
template DNA and primers
.
S
t
ep
s
A
c
ti
on
P
r
ocedu
r
e
de
t
a
il
s
1
Th
aw rea
ge
nts Thaw, mix, and brie
y
centrifu
ge
each co
mp
onent before use
.
2
Pre
p
are 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 rxn
F
in
a
l
co
n
c
.
W
ater, nuclease-
f
re
e
to 50µ
L
5
X
S
up
er
Fi
™
Bu
ff
er
1
1
0µL
1X
1
0
mM d
N
TP mix
1
µL
0
.2mM
e
a
ch
5
X Su
p
er
Fi
™
GC En
h
ancer
(
optiona
l
)
2
1
0
µ
L
1X
Platinum
™
S
uperFi
™
DNA Pol
y
merase
0
.5
µL
0.02U/
L
1
Includes 7.5mM MgC
l
2
.
2
Recommended
f
or targets with >65% GC sequences.
Mix and then brie
y centri
f
uge the components.
3
A
dd tem
p
late DNA and
p
rimers
Add
yo
ur tem
pl
ate DNA and
pr
imers to each tube
f
or a
nal reaction volume o
f
50L.
C
omponen
t
5
0
-µL rxn
Fi
nal conc
.
10µM
f
orward
pr
imer 2.5
µL
0
.5
µM
10µM reverse primer 2.5
µL
0
.5
µM
Te
mp
l
ate DNA
1
va
ri
es
va
ri
es
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n
g
per 50µL reaction. Optimal amount of
ge
nomic DNA is 5–50
ng
per 50µL reaction, but it can
be varied from 0.1n
g
to 250n
g
per 50µL reaction.
C
ap each tube, mix, and then brie
y
centrifu
g
e the content
s.
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).
Mg2+: SuperFi™ Buffer provides MgCl2 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 Mg2+ optimum may be shifted to higher concentrations.
∤If further optimization is needed, increase the Mg2+ 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 Tm calculator available on our website
(www.thermo sher.com/tmcalculator) to calculate the Tm 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 Tm values are at least 69°C
(>20nt) or 72°C (≤20nt), when calculated with our 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.
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 the Tm calculator on our website at www.thermosher.com/tmcalculator to calculate the 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
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1
7
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For Research Use Only. Not for use in diagnostic procedures.
Limited product warranty
Life Technologies Corporation and/or its afliate(s) warrant their products as set forth in the Life Technologies’ General Terms and Conditions of Sale found on Life
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
www.thermosher.com/support.
Disclaimer
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important licensing information
These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label
Licenses.
Corporate Entity: Life Technologies | Carlsbad, CA 92008 USA | Toll free in USA 1.800.955.6288
© 2017 Thermo Fisher Scientic Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientic and its subsidiaries.
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s
s
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Act
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P
rocedure deta
i
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In
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a
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1
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98°
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30 se
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98°
C
30 sec 95°
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5
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Denatur
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98°
C
5
–
1
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9
8°
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9
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7
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1
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MPORTANT! A
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y
s use t
h
e
T
m
ca
l
cu
l
ator on our we
b
site at www.thermosher.com
/
tmcalculator to ca
l
cu
l
ate
yo
ur
p
rimers an
d
t
h
e recommen
d
e
d
annea
l
i
ng
tem
pe
rature
.
Note
:R
efe
r t
o
“
Opt
i
m
i
zat
i
on strateg
i
es
”
, page 1,
f
or guidelines to optimize cycling conditions.
Add
g
el loadin
g
buffer and
a
nalyze w
i
th
g
el
electro
p
hores
i
s
Add gel loading bu
ff
er to 10µL o
f
PCR product, mix, and brie
y centri
f
uge the contents.
Anal
y
ze the sample usin
g
a
g
arose
g
el electrophoresis.
U
se
y
our PCR product immediatel
y
in down-stream applications, or store it at –20°C
.
Troubleshooting
Observation Recommended action
No product at all or low yield. ∤Make sure that there are no pipetting errors.
∤Use fresh high-quality dNTPs. Do not use dNTP mix or primers 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 DNA Polymerase User guide

Brand
Thermo Fisher Scientific
Model
Platinum SuperFi DNA Polymerase
Type
User guide
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