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F1100K

Instruction manual for KOD FX Neo 2004

KOD FX Neo
KFX-201

200 U 200 reactions

Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Quality testing[4]

Primer design[5]

Cloning of PCR products[6]

Protocol
1. Standard reaction setup
2. Cycling conditions[7]

Templates[8]

Examples[9]

Troubleshooting[10]

References[11]

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 FX Neo is based on the DNA polymerase from the hyperthermophilic Archaeon
Thermococcus kodakaraensis KOD11) 2). KOD FX Neo provides greater efficiency andelongation capabilities than conventional PCR enzymes. In particular, KOD FX Neo shows
greater amplification success from crude specimens.
KOD FX Neo is the improved version of the previous KOD FX (Code No. KFX-101).
This product contains a unique “elongation enhancer” that suppresses the “plateau effect”,
enabling greater elongation rates and capabilities.
The KOD FX Neo enzyme solution contains two types of anti-KOD DNA polymerase
antibodies that inhibit the polymerase and 3’5’ exonuclease activities, thus allowing for
Hot Start PCR3). KOD FX Neo generates blunt-end PCR products because of its 3’5’exonuclease (proof-reading) activity.

Features-This enzyme is effective for amplification from crude samples (e.g. biological samples,
foodstuffs, soil extract, etc). Various samples or lysates can be used directly as templates.
-“Elongation enhancer” enables greater amplification efficiency and elongation capability(up to 40 kb from human genomic DNA) compared to conventional PCR. This enzyme isuseful for amplifying difficult targets, such as high G/C, A/T, and/or long targets.

-Various microorganisms (e.g. yeast, fungus, gram-positive bacteria) can be directly usedas templates for PCR.
-The PCR error ratio of KOD FX Neo is about 10 times less than that of Taq DNA
polymerase and is equal to the previous version (KOD FX).

Table 1. Comparison of the mutation frequency of each PCR enzyme.
Fidelity was measured as the mutation frequency by sequencing the PCR product. After cloning the
PCR product (5.7 kb of the human β-globin region), about 96 clones were selected and sequenced.

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

KOD FX Neo (1.0 U/μL) *2× PCR Buffer for KOD FX Neo**
2 mM dNTPs

200 μL × 1
1.7 mL × 3
1 mL × 2*The enzyme solution contains anti-KOD DNA polymerase antibodies that neutralize thepolymerase and 3’5’ exonuclease activities.
** 2× PCR Buffer for KOD FX Neo is a liquid (not frozen) when stored at -20°C. Although
it does freeze below -20°C, the quality is not affected.

[3] Quality Testing

Quality testing is performed by amplification of the human β-globin gene (32 kb).

[4] Primer Design-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 cannot be used.
-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[5] Cloning of PCR
products-KOD FX Neo generates blunt-end PCR products because of its 3’5’ exonuclease (proofreading) activity. Therefore, PCR products can be cloned using blunt-end cloning methods.
-PCR products of KOD FX Neo should 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 [11] Related product).

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Protocol

1. Standard reaction setup
The following protocol is designed for use with the components provided in this kit.
Before preparing the mixture, all components should be completely thawed, except for
the enzyme solution.
Component
PCR grade water

VolumeX μL

Final Concentration
1×

2x PCR buffer for KOD FX Neo

25 μL

1×

2mM dNTPs*

10 μL

0.4 mM each

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

1.5 μL
1.5μL

0.15–0.3 µM
0.15–0.3 µM
Genomic DNA ≤200 ng / 50 μL
Plasmid DNA ≤50 ng / 50 μL
cDNA ≤200 ng （RNA equiv.) / 50 μL
Crude sample ≤0.5–2 μL/50 μL (see [ 7 ])1.0 U / 50 μL

Y μL

Template DNA
KOD FX Neo (1.0U/μL)
Total reaction volume

1 μL
50 μL

* Do not use dNTPs from other kits or companies.
Notes:-Optimal primer concentration is 0.3 µM. In the case of long targets (≥10 kb), a reduced
primer concentration (0.15 µM) may give more effective amplification.
-For PCR reactions, thin-wall tubes are recommended. Reaction setup to a total reaction
volume of 50 μL is also recommended.
-Crude samples (e.g., cultured animal cell suspension) should be added up to 2 μL foreach 50-μL reaction.

2. Cycling conditions
Two-step cycle conditions can be easily used for amplification using KOD FX Neo withprimers ≥20 mer and Tm ≥68°C [recommended cycle]. If the Tm value of the primer isunder 68°C, the 3-step cycle condition is effective. For trouble shooting of poor
amplification (no band, smear etc) or long target amplification (>10 kb), the step-downcycle is recommended. The extension time should be set at 1 min./kb when crude
samples are used as templates.
<Recommended cycle>< 2-step cycle >JAPAN
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Pre-denaturation :For purified DNA/cDNA
94°C, 2 min.

For crude samples
94°C, 2 min.

Denaturation :98°C, 10 sec.

98°C, 10 sec.

Extension :68°C, 30 sec. /kb

68°C, 1 min. /kb

25–45 cycles

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www.toyobo.co.jp/e/bio<Other cycles>< 3-step cycle >Pre-denaturation :For purified DNA/cDNA
94°C, 2 min.

For crude samples
94°C, 2 min.

Denaturation :98°C, 10 sec.

98°C, 10 sec.

Annealing

Tm °C*, 30 sec.

Tm °C, 30 sec.

Extension :68°C,30 sec. /kb

68°C, 1 min. /kb

For crude samples

Pre-denaturation :For purified DNA/cDNA
94°C，2 min.

Denaturation :98°C，10 sec.

98°C，10 sec.

Extension :74°C，30 sec. /kb

74°C，1 min. /kb

Denaturation :98°C，10 sec.

98°C，10 sec.

Extension :72°C，30 sec. /kb

72°C，1 min. /kb

Denaturation :98°C，10 sec.

98°C，10 sec.

Extension :70°C，30 sec. /kb

70°C，1 min. /kb

Denaturation :98°C，10 sec.

98°C，10 sec.

Extension :68°C，30 sec. /kb

68°C，1 min. /kb

Extension :68°C，7 min.

68°C，7 min.

25–45 cycles*Tm value of the primer.

< Step-down cycle >94°C，2 min.
5 cycles5 cycles5 cycles15–30 cycles

Notes:-For amplification from crude samples, the extension time should be 1 min./kb.
-For amplification from a low copy DNA or for longer targets (> 10 kb), longer extension
times may enhance the efficiency.
-Poor amplification may be improved by changing the denaturation step to 94°C, 15 sec.

[7] Templatesa. Purified DNA or cDNA
Appropriate template amounts for 50 μL reaction are summarized in the following table.
Approved range
Recommended
Genomic DNA
Eukaryotic DNA
5–200 ng
50 ng
Prokaryotic DNA
0.1–100 ng
10 ng
Plasmid DNA
10 pg–50 ng
1 ng
cDNA
< 200 ng (RNA equiv.) 50 ng (RNA equiv.)
Lambda phage DNA
0.01–10 ng
1 ng-Contaminating RNA in cDNA or genomic DNA inhibits the PCR reaction by chelating
Mg2+. PCR should be performed using template DNA containing <200 ng RNA.
-Quality of template DNA should be checked by electrophoresis. The length and purityof template DNA affects amplification results.
-Templates containing uracil cannot be used for amplification.

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Tissues and cells

When adding biological samples directly to the PCR reaction solution, the following
samples can be applied to 50 μL reaction.
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
1–2 μL1 × 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 (2–5 μL)

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 to dissociate 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 with KOD FX Neo.

*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 50mM NaOH and vortex.
Spin down.
Incubate at 95°C for 10 min. using 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 an efficient preparation ofmouse tails or nail lysates suitable for amplification with KOD FX Neo. 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 the rapid preparation of a planttissue lysate suitable for amplification with KOD FX Neo.

<Homogenization method>
The following “Homogenization method” is recommended for an effective preparationof a plant tissue lysate suitable for amplification with KOD FX Neo. This method iseffective for the amplification of genomic DNA targets.

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www.toyobo.co.jp/e/bio[8] Examples
Example 1. Amplification from crude samplesa.

Whole blood samples
The human β-globin gene (8.5 kb) was amplified using KOD FX Neo and KOD FX(previous version) with various amounts of blood (EDTA) specimens as templates.
PCR was performed using 50 μL reaction solution contains 1–8 μL whole blood
samples according to the standard protocol with 30 cycles. As a result, KOD FX Neosuccessfully amplified the targets using 1–8 μL whole blood specimens.
Next, the human β-globin gene (1.3 kb) was amplified using various PCR enzymes
with whole blood specimens containing citric acid and heparin as anticoagulants.
KOD FX Neo amplified the targets efficiently from both whole blood samples.

b.

Leaf
Two targets (2.2 and 4.6 kb) were amplified using lysates from tobacco leaves. Each
PCR reaction was performed according to the recommended conditions with 35 cycles.
KOD FX Neo showed greater amplification from lysates prepared by the “one-stepmethod”.
Various targets were then directly amplified using small pieces of tobacco leaves (2× 2 mm). KOD FX Neo successfully amplified DNA using these templates.

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Example 2. Elongation capability
The efficiency for long targets was compared between KOD FX Neo and KOD FX(previous version) using human genomic DNA as templates. The amplification was
performed according to the standard protocol with a step-down cycle. KOD FX Neosuccessfully amplified targets of up to 40 kb.

Example 3. Amplification efficiency and sensitivity
The human β-globin gene (8.5 kb) was amplified using the standard protocol with 40 cycles.
KOD FX Neo showed higher sensitivity than KOD FX (previous version). KOD FX Neo
can suppress the “plateau effect” on PCR after 20 cycles due to the “elongation enhancer”.
Therefore, KOD FX Neo shows greater sensitivity than conventional reagents in the rangeof 30–45 cycles during PCR.

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Example 4. Amplification from mouse tail lysates
The amplification efficiency was then compared between various PCR enzymes using
mouse tail lysates as templates. KOD FX Neo showed greater amplification efficiency thanthe other enzymes.

Example 5. 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.
The result indicates that the error ratio of KOD FX Neo is equal to that of KOD FX(previous version) and approximately 10 times lower than rTaq DNA polymerase.

Table. Comparison of the mutation frequency of each PCR enzyme.

Fidelity was measured as a mutation frequency by sequencing the PCR product. After cloning, the PCR
product (5.7 kb of the human β-globin region), about 96 clones were selected and sequenced.

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Example 6 Extension time for crude samples
Various targets were amplified using crude lysates with two cycling conditions (extension
time: 30 sec./kb or 60 sec./kb). As a result, longer extension conditions gave greater
amplification.

Example 7 Preparation methods of crude lysates
Amplification efficiency was compared using various lysates from mouse tail and tobacco
leaf lysates. For mouse tail lysates, the alkaline lysis method and proteinase K method gavesuperior results. For plant lysates, the one-step and proteinase k methods showed superior
results.

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Application data:

Amplification using soil specimens.

Inhibitory effect of humic acid was compared between various PCR enzymes. Humic acidis the major organic constituent of soil or compost. It is produced by biodegradation ofdead organic matters. The humic acid can be hardly separated from DNA on purificationand inhibits PCR.
The results indicate that KOD FX Neo can amplify targets under high concentrations ofhumic acid.

rDNA was amplified from roughly prepared extracts from 100 mg compost specimens
using the alkaline method [see p5] using various PCR enzymes. In this experiment, three
primer sets for prokaryote, Bacillus sp. and high G/C gram-negative bacteria were used.
The results indicate that KOD FX Neo is the only PCR reagent can amplify all targets.

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

Cause
Cycling condition is notsuitable.

Template DNA is not goodin quality and /or quantity.
No PCR product / low yield

Primer is not good.

Enzyme concentration is
low.
Cycling conditions are notsuitable.

Smearing / Extra band

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

Poor TA cloning efficiency

Too much enzyme
PCR products have bluntends.

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Solution
Increase the extension time up to 60 sec./kb.
Increase the number of cycles by 2–5 cycles.
Use a 3-step cycle instead of a 2-step cycle.
Lower annealing temperature in the 3-step cycling
decrements up to Tm-5–10°C.
Use a step-down cycle.
(This solution is effective for long targets over 10
kb)
Increase the amount of template DNA.
Decrease the amount of template DNA to reducethe contaminated PCR inhibitors.
Use purified templates.
Decrease the amount of cDNA to reduce theinhibition by contaminated RNA.
Degrade or eliminate RNA in the DNA sample.
Decrease the primer concentration from 0.3 mM to
0.15 µM.
(This solution may be effective for theamplification of targets over 10 kb)
Use fresh primers.
Redesign primers.
Increase enzyme concentration to 1.5–2.0 U/ 50
μL .
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 may eliminate smearing or extra
bands)
Reduce Enzyme to 0.5–0.8U/ 50 μL reaction
Clone the PCR products according to general bluntend cloning guide lines.
Use TArget Clone™ -Plus- (Code No. TAK-201)[see related products]

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www.toyobo.co.jp/e/bio[10] References
1)

Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M,
and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997)

2)

Hashimoto H, Nishioka M, Fujiwara S, Takagi M, Imanaka T, Inoue T and Kai Y, J
Mol Biol., 306: 469-77 (2001)

3)

Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126:
762-8 (1999)[ 11 ] Related products
Product name
TArget Clone -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 Clone -Plus- is a highly efficient TA cloning kit. The kit can be applied to the TA
cloning of blunt-ended PCR products amplified using KOD -Plus- [Code No. KOD-201],
KOD -Plus- Neo [Code No. KOD-401], KOD FX [Code No. KFX-101] or KOD FX Neo[Code No. KFX-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 from KOD -Plus[Code No. KOD-201], KOD -Plus- Neo [Code No. KOD-401], KOD FX [Code No. KFX101] and KOD FX Neo [Code No. KFX-201]. possess blunt ends due to 3’5’ exonucleaseactivity 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 T-vectors 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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