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Instruction manual for KOD OneTM PCR Master Mix / KOD OneTM PCR Master Mix -Blue- 2004

F1696K

KOD OneTM PCR Master Mix
KOD OneTM PCR Master Mix -BlueKMM-101
KMM-201

1 mL x 5
1 mL x 5

Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Quality testing[4]

Primer design[5]

Protocol
1. Standard reaction setup
2. Cycling conditions[6]

Templates[7]

Cloning of PCR products[8]

Examples[9]

Troubleshooting[10]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory
precautions and safety while using this kit.

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www.toyobo.co.jp/e/bio[1] Introduction

Description
KOD OneTM PCR master Mix and KOD OneTM PCR Master Mix -Blue- are 2 x PCR
master mixes based on genetically modified KOD DNA polymerase (UKOD). KOD OneTM
series enables fast PCR, which has an extension time of 5 sec./ kb by applying UKOD anda new Elongation Accelerator. In addition, these master mixes provide greater efficiency
and elongation capabilities than conventional PCR enzymes. In particular, these show
greater amplification success from crude specimens. Furthermore, these master mixes canbe applied to amplify from templates containing uracils (dU) or using primers containing
inosines (dI) and uracils (dU).
KOD OneTM series contains two types of anti-KOD DNA polymerase antibodies that
inhibit the polymerase and 3’5’ exonuclease activities, thus allowing for Hot Start PCR.
These master mixes generate blunt-end PCR products because of 3’5’ exonuclease(proof-reading) activity of KOD DNA polymerase.

Features- Fast
KOD OneTM series can amplify the targets using the following very short conditions:≤ 1 kb:1 sec.
1~ 10 kb: 5 sec./ kb
10 kb~:
10 sec./ kb
The cycling conditions can be set flexibly when various targets having different sizes areamplified.
- Easy to Use
KOD OneTM series contains all reaction components except primers and templates andprovide high reproducibility by reducing operations. In addition, KOD OneTM PCR Master
Mix -Blue- includes a loading dye (BPB) to allow direct loading onto agarose gels.
- High Fidelity
KOD OneTM series exhibits approximately 80-fold higher fidelity than Taq DNA
polymerase. These mixes can be used for various purposes where this would be anadvantage (e.g., in the preparation of long target amplicons for sequencing).
- High Efficiency
KOD OneTM series is effective for amplification from crude samples (e.g., biologicalsamples, foodstuffs, soil extract, etc.). Various samples or lysates can be used directly as
templates.
- Primers or Templates Containing Inosines (dI) or Uracils (dU) Can Be Used
KOD OneTM series can use primers or templates containing inosines (dI) or uracils (dU),
whereas conventional high-fidelity PCR enzymes cannot.

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www.toyobo.co.jp/e/bio[2] Components

KOD OneTM series includes the following components for 200 reactions, 50 μl total
reaction volume.
<KMM-101>
KOD OneTM PCR master Mix

1 mL x 5<KMM-201>
KOD OneTM PCR master Mix -Blue-

1 mL x 5

Note:
The reagents can be stored at 4°C for a month. For longer storage, the reagents should be
kept at -20°C.

[3] Quality Testing[4] Primer Design

Quality testing is performed by amplification of a 10 kb target using fast PCR, which has
an extension time of 50 sec.
Technical information (data) contained in this instructional manual are for description
representation and application of the product and does not constitute any guarantee.

-Primers should be 22–35 bases with a melting temperature (Tm) over 63°C.
-Optimal GC content of primers is 45%–60%. The ideal GC contents of the 5’ half and the
3’ half are 60%–70% and 45%–50%, respectively.
-The priming efficiency of primers can be promoted by anchoring the 3’end of primers
with G or C.
-Primers should be designed so as not to generate intermolecular secondary structures orprimer dimers.
-Primers for long target amplification should be 25–35 bases with Tm over 65°C.
-Primers containing inosine (dI) or uracil (dU) can be used and are applicable for many
purposes such as metagenomics analyses. The design of primers can be facilitated by useof specific software that allow replacement of thymine (dT) with uracil (dU) or inosine(dI).
The following online tool is recommended for amplification after bisulfate.
MethPrimer, http://www.urogene.org/methprimer/index.html-The Tm of primers should be calculated using the Nearest Neighbor method. The Tm
values in this manual were calculated using this method with the following parameters.
Na+ concentration: 50 mM
Oligonucleotide concentration: 0.5 µM

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Protocol

1. Standard reaction setup
Before preparing the mixture, all components should be completely thawed, except for
the enzyme solution.
Component
PCR grade water

VolumeX μL

Final Concentration

KOD OneTM PCR Master Mix

25 μL

1×

10 pmol /μL Primer #1
10 pmol /μL Primer #2

1.5 μL
1.5 μL0.3 µM
0.3 µM
Genomic DNA ≤200 ng / 50 μL
Plasmid DNA ≤50 ng / 50 μL
cDNA ≤750 ng （RNA equiv.) / 50 μL
Crude sample ≤5 μL /50 μL

Y μL

Template DNA
Total reaction volume

50 μL

Notes:-Optimal primer concentration is 0.3 µM. In the case of long targets (≥10 kb), reduced
primers concentration (0.15 µM) may give more effective amplification.
-When PCR yield is low, increased primers concentration (0.5 µM) may give more
effective amplification.
-For PCR reactions, thin-walled tubes are recommended.

2. Cycling conditions
The following cycle is recommended.
3-step cycle
Denaturation :Amplicon size
≤ 1 kb
98°C, 10 sec.

Amplicon size
1 ~10 kb
98°C, 10 sec.

Amplicon size
≥ 10 kb
98°C, 10 sec.

Annealing :(Tm -5) °C, 5 sec.

(Tm -5) °C, 5 sec.

(Tm -5) °C, 5 sec.

Extension :68°C, 1 sec.

68°C, 5 sec. /kb

68°C, 10 sec. /kb

Notes:-Longer extension time may enhance efficiency. For amplification from a low copy
DNA or crude sample, the extension time should be 10 sec. /kb.
-The extension temperature should be adjusted in accordance with the Tm of primers.
If the Tm value of the primer is over 73°C, the extension temperature should be set at
68°C.
-Poor amplification may be improved by changing the denaturation step to 94°C, 15 sec.

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25–45 cycles

www.toyobo.co.jp/e/bio<Other cycles>
When non-specific or smeared bands are observed, the following cycles isrecommended.
3-step cycle[6] Template

Denaturation :Amplicon size
< 10 kb
98°C, 10 sec.

Amplicon size
≥ 10 kb
98°C, 10 sec.

Extension :68°C, 5 sec. /kb

68°C, 10 sec. /kb

Step-down cycle
Denaturation :Amplicon size
< 10 kb
98°C，10 sec.

Amplicon size
≥ 10 kb
98°C，10 sec.

Extension :74°C，5 sec. /kb

74°C，10 sec. /kb

Denaturation :98°C，10 sec.

98°C，10 sec.

Extension :72°C，5 sec. /kb

72°C，10 sec. /kb

Denaturation :98°C，10 sec.

98°C，10 sec.

Extension :70°C，5 sec. /kb

70°C，10 sec. /kb

Denaturation
15–30
cycles :Extension :98°C，10 sec.

98°C，10 sec.

68°C，5 sec. /kb

68°C，10 sec. /kb

25–45 cycles

5 cycles5 cycles5 cyclesa. Purified DNA or cDNA
Appropriate template amounts for a 50 μL reaction are summarized in the following table.
Approved range
Recommended
Genomic DNA
Eukaryotic DNA
1–200 ng
50 ng
Prokaryotic DNA
0.1–200 ng
10 ng
Plasmid DNA
1 pg–50 ng
10 ng
cDNA
< 750 ng (RNA equiv.) 50 ng (RNA equiv.)
Lambda phage DNA
0.01–10 ng
1 ng-Contaminating RNA in cDNA inhibits the PCR reaction. PCR should be performed
using template DNA containing <750 ng of RNA in a 50 μl reaction.
-Quality of the template DNA should be checked by electrophoresis. The length andpurity of the template DNA affects amplification results.
b.

Tissues and cells

When adding biological samples directly to the PCR reaction solution, the following
samples can be applied to the 50 μL reaction.

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SampleE. coli
Yeast
FungusAppropriate template amount
Picked small amount of cells from colonies
Picked small amount of cells from colonies
Picked small amount of cells from colonies

Cultured cells
Whole Blood
Nail
Hair root
Leaf
Milled rice
Mouse tail101–105 cells/2μL medium or PBS
1–2μL
1 × 1 mm
1–2 cm2 × 2 mm
0.5 × 0.5 mm
1 × 1 mm

Remarks

When reproducibility is not
good, suspended cells in TE
buffer should be added (2–5μL)

As the concentration of extracted
DNA is low, 35–40 cycles are needed.

On an agarose gel assay, a portionof amplicon may remain in the slots.
*In the case of the direct amplification from animal tissues, such as mouse tail, a portionof the amplification product may remain in the gel slot on an agarose gel assay. Adding 10
μL of 20 mg/mL proteinase K to 50 μL PCR products prior to the electrophoresis iseffective for dissociating the aggregates.
c.

LysateTo make the lysate for PCR, the following methods are recommended. The lysates can be
stored at 4°C for several weeks. For long-term storage, the lysates should be stored at 20°C.
<Alkaline lysis method>
The following “Alkaline lysis method” is recommended for rapid preparation of mouse
tails or nail lysates suitable for amplification.

*Mouse tail cannot be dissolved completely.

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www.toyobo.co.jp/e/bio[96-well PCR plate protocol by alkaline lysis method]
The following protocol is useful for the preparation of lysates from a large number ofmouse tail samples.
1.
2.
3.
4.
5.
6.

Transfer mouse tails (ca 3mm) to a 96-well PCR plate.
Add 180 μL of 50 mM NaOH and vortex.
Spin down.
Incubate at 95°C for 10 min using a thermal cycler.
Add 20 μL of 1M Tris-HCl (pH 8.0) and vortex.
Spin down.

Supernatant 0.5–2 μL 

PCR (50 μL)<Proteinase K method>
The following “Proteinase K method” is recommended for efficient preparation ofmouse tails or nail lysates suitable for amplification. This protocol can also be applied
to the following samples.
Mouse tail: 3 mm
Nail: 3 mm
Leaf: 3 × 3 mm
A rice grain

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www.toyobo.co.jp/e/bio<One step method>
The following “One-step method” is recommended for rapid preparation of a planttissue lysate suitable for amplification.

<Homogenization method>
The following “Homogenization method” is recommended for effective preparation ofa plant tissue lysate suitable for amplification. This method is effective for amplificationof genomic DNA targets.

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www.toyobo.co.jp/e/bio[7] Cloning of PCR
products-KOD OneTM PCR Master Mix / KOD OneTM PCR Master Mix -Blue- generate blunt-end
PCR products because of 3’5’ exonuclease (proof-reading) activity. Therefore, PCR
products can be cloned using blunt-end cloning methods.
-PCR products of KOD OneTM PCR Master Mix / KOD OneTM PCR Master Mix -Blueshould be purified prior to restriction enzyme treatments in cloning steps. The 3’5’exonuclease activity of KOD DNA polymerase remains at the end of the PCR reaction.
-The dedicated TA cloning kit “Target Clone™ -Plus- (Code No. TAK-201)” isrecommended for the cloning of blunt-end PCR products produced by KOD DNA
polymerase (see [10] Related product).

[8] Examples

Example 1. Fast PCR
Various targets were amplified with KOD OneTM PCR Master Mix and KOD OneTM PCR
Master Mix -Blue- using the fast cycling conditions. KOD OneTM series successfully
amplified all targets.
<Reaction Mix>
PCR grade water
KOD OneTM PCR Master Mix
10 μM Primers
10 ng/ μL human genomic DNA
Total Volume

21 μL
25 μL
1.5 μL
1 μL
50 μL<PCR cycle>
Amplicon size ≤ 1 kb
98℃ 10 sec.
60℃ 5 sec.
×30 cycles
68℃ 1 sec.
Amplicon size 1 ~10 kb
98℃ 10 sec.
60℃ 5 sec.
×30 cycles
68℃ 5 sec.

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Example 2. PCR error ratio
The error ratio of various PCR enzymes were compared by determining the sequences of
the amplicons from human β-globin gene. The amplicons were cloned into the vector using
TArget clone™ -Plus- (Code No. TAK-201) and the sequences were determined.
KOD OneTM PCR Master Mix and KOD OneTM PCR Master Mix -Blue- showed excellent
fidelity and the mutation frequency was approximately 80 times lower than that of rTaq
DNA polymerase.

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Example 3. Amplification efficiency and sensitivity
The 3.5 kb fragments were amplified from human genomic DNA, cDNA and plasmid DNA.
Each PCR reaction was performed according to the recommended conditions. KOD OneTM
PCR Master Mix showed higher sensitivity using 5 sec./ kb extension time.
<Reaction Mix>
PCR grade water
KOD OneTM PCR Master Mix
10 μM Primers
Each template DNA
Total Volume

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21 μL
25 μL
1.5 μL
1 μL
50 μL<PCR cycle>
98℃ 10 sec.
60℃ 5 sec.
68℃ 18 sec.

×30 cycles

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Example 4. Amplification from cDNA
The inhibitory effect of RNA in cDNA was compared using various PCR enzymes. KOD
OneTM Master Mix was not susceptible to RNA inhibition, and it was able to amplify targets
under high concentrations of cDNA.
<Reaction Mix>
PCR grade water
KOD OneTM PCR Master Mix
10 μM Primers
Each cDNA
Total Volume

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17 μL
25 μL
1.5 μL
5 μL
50 μL<PCR cycle>
98℃ 10 sec.
60℃ 5 sec.
68℃ 18 sec.

×30 cycles

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Example 5. Amplification from crude samples
Amplification from whole blood and that from mouse lysate were compared. KOD OneTM
PCR Master Mix amplified the targets efficiently.
<Reaction Mix>
PCR grade water
22 - X μL
KOD OneTM PCR Master Mix 25 μL
10 μM Primers
1.5 μL
Each template
X μL
Total Volume
50 μl<PCR cycle>
98℃ 10 sec.
60℃ 5 sec.
68℃ 5 sec./ kb

×30 cycles

Example 6. Amplification using degenerate primers containing inosine.
The 2.8 kb fragments were amplified using degenerate primers containing inosine. KOD
OneTM PCR Master Mix was able to amplified, whereas conventional high-fidelity PCR
enzymes were not.
<Reaction Mix>
PCR grade water
KOD OneTM PCR Master Mix
10 μM Primers*
50 ng/μl E.coli genomic DNA
Total Volume

21 μL
25 μL
1.5 μL
1 μL
50 μL<PCR cycle>
98℃ 10 sec.
60℃ 5 sec.
68℃ 15 sec.

×30 cycles<Primer sequence>
Fwd: ATGGTICARATHCCICARAAY
Rev: RTGIGCYTGRTCCCARTTYTC*In cases where degenerate primers are used, the primer
concentrations should be increased depending on the degree ofdegeneracy.

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www.toyobo.co.jp/e/bio[9] Troubleshooting
Symptom

Cause
Cycling condition is
unsuitable.

Template DNA is not goodin quality and /or quantity.

No PCR product / low yield
Primer is not good.

Cycling conditions are
unsuitable.

Smearing / Extra band

Too much template DNA.
Quality of primers is not
sufficient.

Poor TA cloning efficiency

Too much enzyme
PCR products have blunt
ends.

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Solution
Increase the extension time up to 10 ~30 sec./ kb.
Increase the number of cycles by 2–5 cycles.
Lower annealing temperature in the 3-step cycle
decrements up to Tm-7–10°C.
Increase the amount of template DNA.
Decrease the amount of template DNA to reducethe contaminated PCR inhibitors.
Use purified templates.
Degrade or eliminate RNA in the DNA sample.
Decrease the primer concentration from 0.3 µM to
0.15 µM. This solution may be especially effective
for the amplification of targets over 10 kb.
Increase the primer concentration from 0.3 µM to
0.5 µM. This solution may be especially effective
for amplification from low copies template.
Use fresh primers.
Redesign primers.
Change from 3-step cycling to 2-step cycling.
Change from 2-step cycling to step-down cycling.
Decrease the number of cycles by 2–5 cycles.
Reduce the amount of template DNA.
Use fresh primers.
Redesign primers.
(Longer primers (25-35 mer) may eliminate
smearing or extra bands)
Reduce enzyme to 0.5–0.8U/ 50 μL reaction.
Clone the PCR products according to general
Follow blunt-end cloning guidelines.
Use TArget Clone™ -Plus- (Code No. TAK-201)[see [10] Related products].

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www.toyobo.co.jp/e/bio[10] Related products
Product name
TArget CloneTM -Plus10× A-attachment mix
Ligation high Ver.2

Package
10 reactions
25 reactions
750 μL(100 reactions)

Code No.
TAK-201
TAK-301
LGK-201

TArget CloneTM -Plus- is a highly efficient TA cloning kit. The kit can be applied to the
TA cloning of blunt-ended PCR products amplified using products containing KOD DNA
polymerase. (KOD -Plus- [Code No. KOD-201], KOD -Plus- Neo [Code No. KOD-401],
KOD FX [Code No. KFX-101], KOD FX Neo [Code No. KFX-201], KOD -Multi & Epi[Code No. KME-101] or KOD OneTM PCR Master Mix [Code No. KMM-101, KMM-201].
The kit contains pTA2 Vector, 2x Ligation Buffer, T4 DNA Ligase, and 10× A-attachment
Mix.
10× A-attachment mix is a reagent comprising anti-KOD DNA polymerase antibody
specific to KOD 3’5’ exonuclease activity (proof-reading activity), as well as Taq DNA
polymerase, which exhibits terminal transferase activity. PCR products using KOD DNA
polymerase possess blunt ends due to 3’5’ exonuclease activity of the KOD DNA
polymerase. The 10× A-attachment mix allows for PCR products to acquire overhanging
dA at the 3’ ends. Products with 3’-dA overhangs can be directly cloned into arbitrary Tvectors using ligation reagents, such as Ligation high Ver.2 [Code No. LGK-201].

Figure.

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Principle of the 10× A-attachment mix

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