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F0936K

Instruction manual KOD -Plus- Mutagenesis Kit 2004

KOD -Plus- Mutagenesis Kit
SMK-101

20 reactions

Store at -20°C

Contents[1]

Introduction[2]

Flow chart[3]

Components

[4]

Notes

[5]

Protocol1.

Inverse PCR

2. Dpn I Digestion of the Template Plasmid
3. Self-ligation of the PCR Product4. Transformation
5. Analysis of Transfomants[6] Troubleshooting[7] References

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe generallaboratory precaution and utilize safety while using this kit.

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

Description
This kit is an inverse PCR (iPCR)-based site-directed mutagenesis kit using KOD DNA
polymerase1) 2) as a high-fidelity PCR enzyme. This reagent was developed based on a highfidelity and efficient PCR reagent, “KOD-Plus- (Code No. KOD-201)”, which consists of
KOD DNA polymerase and anti-KOD DNA polymerase antibodies3) for Hot Start PCR.
This kit enables not only the introduction of point mutations, but also the introduction oflarge insertions and deletions. The PCR fidelity of KOD-Plus- is greater than Taq DNA
polymerase (ca. 80-fold); therefore, unexpected, 2nd-site mutations can be reduced. PCR
reactions can be performed using standard PCR primers and do not require phosphorylated
primers, because this protocol contains a ‘Phosphorylation Step’ of PCR products.

Features-Applicable for various mutations, such as substitutions, insertions, anddeletion mutations.
-High efficiency (95% maximum) can be obtained.
-Simple protocol facilitates speedy experiments. Phosphorylated primers are
not required.

[ 2 ] Flow chart

The flow chart for this kit is as follows:

PCR Primerx Mutation
CH3 Methylated site

CH3 CH3Template CH3 CH3(Plasmid)

CH3CH3Inverse PCR （5～10 cycles ）＜0.5～2hr＞

ACH3CH3CH3(A) Inverse PCR of plasmid DNA,using a mutation primer.
(B) Plasmid DNA is digested by DpnI.

CH3Digestion of the templateplasmid by Dpn I ＜37℃ 1hr＞

Note: Dpn I digests methylatedDNA, such as plasmid DNA fromtypical E. coli cell lines (e.g.
JM109 and DH5α).

PCR products are not digested by Dpn I.
※DpnI digests methylated sites.

(C) Self-ligation of PCR products is
performed by a reaction with T4
polynucleotide kinase and ligase.

PCR products

CH3Template
CH3

BC

Self-ligation of PCR products（Kinase / Ligase）＜16℃ 1hr＞(D) Transformation of E. coli cells
using self-ligated PCR products.

MutantDTransform E.coli

Figure 1. Flowof KOD -Plus- Mutagenesis Kit[ 3 ] chartComponents
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www.toyobo.co.jp/e/bio[ 3 ] Components

The KOD -Plus- Mutagenesis Kit contains enough reagents for 20 mutagenesis reactions,including 5 control reactions.
KOD-Plus-（1 U/μL）
10x Buffer for iPCR
2 mM dNTPs
DpnI (10 U/μL)
T4 Polynucleotide Kinase (5 U/μL)
Ligation high (T4 Ligase + Buffer Mixture)
Control Plasmid pAK119M (50 ng/μL)
Control Primer #1 (10 pmol/μL)
Control Primer #2 (10 pmol/μL)

25 μL
125 μL125 μL
50 μL
50 μL
250 μL
10 μL
10 μL
10 μL

＜Additional Materials Required ＞
・Target plasmid DNA (plasmid DNA should be isolated from commonly used E.coli
strains that bear dam-methylase, such as JM109 and DH5α.)・Mutagenic primers (normal primers, not phosphorylated primers, should be used.)・Competent cells
・SOC medium and LB agar plates with antibiotics
・4% X-Gal and 100 mM IPTG (for control reaction)

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www.toyobo.co.jp/e/bio[ 4 ] Notes
1. Template plasmids
In this kit, template plasmids are digested through treatment with the restriction
enzyme Dpn I. The template plasmid should be methylated, since Dpn I recognizes
the Gm6ATC (m6 methylated) site. The “GATC” recognition sites of plasmid DNA
purified from ordinary host cells (e.g., E. coli JM109 or DH5α) are m6-methylated by
the host cell’s Dam methylase.

2.

PCR primers(1) Primer design
Mutation sites should be designed at the 5’ terminal end of primers. Specific sites
should be designed at the 3’ region of the primers. The optimal length of the specificregions should be ≥ 20 mer (preferably 25 mer).
This kit includes a kination step of the PCR product; therefore, phosphorylated
primers are not required.
(2) Quality of primers
Primer quality is critical for obtaining good results. HPLC-grade primers, which do
not contain deficient regions at the 5’ termini, are suitable for this experiment. In thecase of long primers (≥ 40 mer), PAGE-grade primers are recommended.

Substitution

Deletion

InsertionorTemplate
plasmid

Figure 2. Examples of primer design for site-specific mutations-Substitution: Mutations should be introduced around the 5’ termini of primers.
Deletion: The deletion site should be introduced between the 5’ termini of primers.
Insertion: The insertion site should be introduced at the 5’ termini of primers.
In the case of a broad insertion, the insertion region can be divided into 2 parts and introduced
at the 5’ termini of the forward and reverse primers.

3.

PCR conditions

Specific amplification of the target sequence is critical to obtain the expected mutant clones.
Consequently, a preliminary amplification test with 10~20 cycles can be helpful to confirm
PCR specificity. If specific amplification is obtained, the same cycling conditions can beutilized with fewer cycles.

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4. Unexpected mutations (2nd mutation)
KOD DNA polymerase exhibits excellent accuracy in PCR. However, the introduction ofunexpected mutations in other primer regions cannot be excluded. Mutants should be
confirmed by sequencing.

5. Control plasmid and control reaction
This kit provides a control plasmid (pAK119M), bearing the mutated LacZα gene. The
6th codon has been changed from CCA to TAA (stop codon). Because the mutated LacZαgene exhibits no activity, E. coli colonies expressing this gene form white colonies on Xgal agarose plates. A control reaction can be performed using this plasmid and mutation
primers. This reaction reverts the stop codon (TAA) to the wild-type codon (CCA).
The control reaction flow chart using the control plasmid provided in this kit as follows:pAK119M
4.3 kblacZαTAA（stop）…White ColonySite-directed Mutagenesis using Control Primers
CCA（Pro）…Blue Colony

LacZαN…MetThrMetIleThrSTOP…C
5’…AATTTCACACAGGAAACAGCTATGACCATGATTACGTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATC…3’3’…TTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCATTCGAACGTACGGACGTCCAGCTGAGATCTCCTAG…5’Control Primer #15’…CCAAGCTTGCATGCCTGCAGGTCGA…3’5’…AATTTCACACAGGAAACAGCTATGACCATGATTACGTAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATC…3’3’…TTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCATTCGAACGTACGGACGTCCAGCTGAGATCTCCTAG…5’3’…CCTTTGTCGATACTGGTACTAATGC…5’Control Primer #2

N…MetThrMetIleThrProSerLeuHisAlaCysArgSerThrLeuGluAsp…C
5’…AATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGAT…3’3’…TTAAAGTGTGTCCTTTGTCGATACTGGTACTAATGCGGTTCGAACGTACGGACGTCCAGCTGAGATCTCCTA…5’Figure 3. Flow chart of the control experiment.

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www.toyobo.co.jp/e/bio[ 5 ] Protocol

1.

Inverse PCR(1) Prepare PCR primers (10 pmol/μL) and template plasmid (50 ng/μL).
(2) Prepare the PCR reaction mixture as follows:
PCR grade water

35 μL

10×Buffer for iPCR

5 μL

2 mM dNTPs

5 μL

Primer A（10 pmol/μL）

1.5 μL

Primer B（10 pmol/μL）

1.5 μL

Plasmid Template DNA（50 ng/μL）

1 μLKOD -Plus- DNA Polymerase

1 μL

Total Volume

50 μL(3) Perform PCR reaction as follows:
94°C
98°C
68°C
4°C

2 min.
10 sec.
X min.*1HoldY（4~10）Cycles＊2*1 Extension time should be 1 min/kb of plasmid length (e.g., a 5 kb-size

plasmid: 5 min.).
*2 Cycle number should be 1 cycle/kb of plasmid length (e.g., a 5 kb-size

plasmid: 5 cycles).
Notes
If the Tm value of the specific regions of the primers are less than 73°C,
the following 3 step condition is recommended;
94°C
98°C
Tm*1-[5~10] °C
68°C
4°C

2 min.
10 sec.
30 sec.
X min.*２HoldY（4~10）Cycles＊３*1 Tm value of the specific region of the primer.
*2 Extension time should be 1 min./kb of plasmid length (e.g., a 5 kb-size

plasmid: 5 min.).
*3 Cycle number should be 1 cycle/kb of plasmid length (e.g., a 5 kb-size

plasmid: 5 cycles).

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www.toyobo.co.jp/e/bio(4) [Optional] The control reaction is as follows:
PCR grade water

35 μL

10×Buffer for iPCR

5 μL
5 μL

2 mM dNTPs
Control Primer #1（10 pmol/μL）

1.5 μL

Control Primer #2（10 pmol/μL）

1.5 μL

Control Plasmid pAK119M （50 ng/μL）

1 μL

KOD -Plus- (1 U/μL

1 μL

Total Volume

50 μL

94°C
98°C
68°C

2.

2 min.
10 sec.
5 min.

5 cyclesDpnI digestion of the template plasmid(1) Add 2 μL of DpnI restriction enzyme (10 U/μL) to 50 μL PCR reaction fromstep 1.
Notes
Add 1 μL of the DpnI restriction enzymes to 25 μL PCR reaction. (half ofreaction volume)(2) Gently and thoroughly mix each reaction mixture by pipetting.
(3) Spin down the reaction mixture and incubate at 37°C for 1 hr to digest thetemplate plasmid.
（Optional） Gel electrophoresis
To confirm PCR amplification, we recommend agarose gel electrophoresis analysis.
However, in the case of 5 or less cycles of amplification, it may be only possible todetect a faint band or no band at all. If only a faint or no band is detected, pleaseproceed to the next step.

3.

Self-ligation of the PCR Product(1) Prepare the kination/ligation reaction mixture using a fresh tube as follows:

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DpnI- treated PCR product

2 μL

PCR grade water

7 μL

Ligation high

5 μL

T4 Polynucleotide Kinase

1 μL

Total Volume

15 μL

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www.toyobo.co.jp/e/bio(2) Gently and thoroughly mix each reaction mixture by pipetting.
(3) Centrifuge the reaction mixture and incubate each reaction at 16°C for 1 hr.

4.

Transformation
Standard E. coli strains can be used as the transformation host. Bothelectrocompetent and chemically competent E. coli cells can be used. The following
protocol is a standard protocol using chemically prepared competent E. coli cells.
(1) Gently thaw the competent cells （100 μL） on ice.
(2) Add 10 μL of reaction mixture from Step 3 to the competent cells, swirl gentlyand incubate for 30 min. on ice.
NoteTo verify transformation efficiency of the competent cells, 1 pg ofpBR322 can be used as a control plasmid.
(3) Incubate for 30 seconds at 42°C and then place on ice for 2 min.
(4) Add 900 μｌ of SOC medium and incubate at 37°C for 1 hr with shaking.
(5) Plate appropriate volumes (10~200 μL) of transformed E. coli solution on agar
plates containing the appropriate antibiotic for the plasmid vector.
Note
For the mutagenesis controls, spread cells onto LB-Ampicillin (or LBKanamycin) agar plates containing X-gal and IPTG.
(6) Incubate the plates at 37°C for ~16 hr.
NoteThe expected colony number from the transformation of the pAK119M
control mutagenesis reaction is between 50 and 500 when competentcells with transformation efficiency 1x109 cfu/µg-pBR322 are used.
Greater than 80% of the colonies should contain the mutation andappear as a blue colony on agar plates containing X-gal and IPTG.

5.

Analysis of Transfomants
A typical mutagenesis efficiency of this kit is greater than 80%. Therefore, analysisof 4-8 clones is typically appropriate for sequence verification.

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www.toyobo.co.jp/e/bio[ 6 ] Troubleshooting
Symptom
Low colony
numberExcess colony
numberNo mutant

Failure of thecontrol experiment[ 7 ] References

CauseSolution

Error of Inverse PCR
reaction

Reconfirm the primer design and PCR conditions.

Cycle number is not
sufficient

Increase the cycle number up to 10 cycles.

Transformation efficiency ofthe competent cells is low

Plasmids are notmethylated

Check the transformation efficiency of the competentcells using a standard plasmid such as pBR322.
Competent cells showing >108 cfu/µg-pBR322 aresuitable.
Use E. coli host bearing dam-methylase for the templateplasmid preparation, such as JM109 or DH5α.

Concentration ofantibiotics is not appropriate

Use the appropriate concentration of antibiotics. Ensure
antibiotics are fresh.

Error of Inverse PCR

Reconfirm the primer design and PCR conditions.

Plasmids are notmethylated

Use E. coli host bearing dam-methylase for the templateplasmid preparation, such as JM109 or DH5α.

Concentration of IPTG orX-gal is not correct.

Confirm the concentration of IPTG or X-gal.

Host E. coli cell is not
appropriate.

Use E. coli strains suitable for white/blue selection suchas DH5α or JM109.

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, JMol Biol., 306: 469-77 (2001)

3)

Mizuguchi H, Nakatsuji M, Fujiwara S, Takagi M and Imanaka T, J Biochem., 126:
762-8 (1999)JAPANTOYOBO CO., LTD.
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