www.toyobo.co.jp/e/bio

F1016K

Instruction manual Ligation high Ver.2 2004

Ligation high Ver.2

LGK-201

750 μL(100 reactions)Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Protocol[4]

Application data
Example 1. Cohesive end ligation
Example 2. Blunt end ligation
Example 3. Linker ligation
Example 4. TA cloning using purified PCR products
Example 5. TA Cloning using unpurified PCR products
Example 6. Phage vector ligation[5]

Troubleshooting

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

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

NOT FOR HUMAN OR DIAGNOSTIC USE.

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

Description
The ligation reaction is an essential step in genetic recombination experiments. For thisreaction, T4 DNA ligase has been widely used. Ligation high Ver.2 is a highly efficient
premixed T4 Ligase reagent.

Features
Relative colony number- Effective ligation of cohesive, blunt and A overhang DNA fragments can be achieved.
120
100
80BlueWhite

60
40
20itor

itor
Com

Com

pet

pet

Ve
high
ion
Ligat

Fig. 1

BAr.2

0Comparison of TA cloning efficiency

Colony number of Ligation high Ver.2 corresponds to 100- Will not freeze at -20℃. No need to thaw.
- Just mix ligation high Ver.2 with an equal volume or with double the volume of thesolution containing DNA fragments.

[ 2 ] Components

750 μL ×

Ligation high Ver.2

1Vial(100 reactions)

Relative colony numberNotes:
In the case of -30℃ storage or long-term storage under -20℃, the reagent might freeze orform white precipitates. The frozen reagent can be thawed by incubation at room
temperature in short time. No decrease in reaction efficiency is observed following 5
freeze-thaw cycles. The white precipitates should be dissolved prior to use.The white
precipitates can also be dissolved by incubation at room temperature with vortexing at low
speed in short time.
12010080BlueWhite60
40
20005Times of Freezing and thawFig. 2 Effect of freezing and thaw for activity of Ligation high Ver.2
Ligation efficiency was estimated by the TA cloning test.
Colony number at 0 times of freezing and thaw corresponds to 100.
No negative effect was detected by 5-time-freezing and thaw.
JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

1NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 3 ] Protocol
7.5 μL

Vector DNA＋Insert DNA*1

3.75-7.5 μL *2

Ligation high Ver.2
16℃、30 min.*3*4↓

Transform 100 μL competent cells using 1 -10 μl reaction solution *5*1 Ligation efficiency is decreased by adding excess salts. DNA fragments should be inlow salt solutions (e.g. 10 mM Tris-HCl [pH 8.0], 1 mM EDTA)*2 Adding an equal volume of Ligation high Ver.2 to the DNA solution is recommended in
the case of TA cloning.
*3 The reaction can be prolonged for up to 2 hr. Standard cohesive end ligations can becompleted within 5 min.
*4 The reaction can be performed at 4℃-25℃.
*5 The volume of ligation reaction added should be less than 10% of the volume of thecompetent cell suspension. For electro-transformation, ligation reactions should bepurified, because salts inhibit the transformation.
Table 1

Recommended reaction conditions
DNA： Ligation reagent

Reaction condition(Volume)Cohesive end ligation

1 ： 1or1 ： 0.5

16℃、5-30 min.

Blunt end ligation

1 ： 1or1 ： 0.5

16℃、30 min.

Linker ligation1 ： 1or

1 ： 0.5

16℃、30 min.

T vector ligation

1 ： 116℃、30 min.

1 ： 116℃、30 min.

（Purified PCR procuct）
T vector ligation *1*2
（Unpurified PCR product）
Phage vector ligation
1 ： 1 or 1 ： 0.5
16℃、30 min.
*1 When PCR reactions contain primer dimers or unexpected PCR products, the desired
products should be purified.
*2 When sufficient amplification is confirmed, 0.5-1 μL of PCR product can be useddirectly. The reaction conditions are as follows;
H20PCR product
Ligation high Ver.2

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

X0.5-17.5
15 (μL)CHINATOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

2NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 4 ] Application data

Example 1. Cohesive end ligation

120
100
80
60
40
20
01：
51：
11：
01：
3Blue
White.5

Relative colony number

Dephosphorylated pUC19/HindIII,（50 ng, 25 fmol,）was mixed, at different ratios, withthe 546 bp DNA fragment of HindIII digested phage lambda DNA. An equal volume ofLigation high Ver.2, 7.5 μL, was added and incubated at 16℃ for 30 min. E. coli DH5
α competent cells were transformed using 10 μL of reaction mixture, and cultured on
LB/Amp (X-Gal) plates O/N.

Vector ： Insert （molar ratio）

Fig. 3 Cohesive end ligation
Colony number at 1:5 corresponds to 100

Example 2.

Blunt end ligation

Dephosphorylated pUC19/HincII,（50 ng, 25 fmol）was mixed, at different ratios, withpurified PCR product (500 bp: from high-fidelity PCR enzyme*1). Half or an equalvolume of Ligation high Ver.2, 3.75 or 7.5 μL, was added and incubated at 16℃ for 30
min. E. coli DH5α competent cells were transformed using 10 μL of reaction mixture,and cultured on LB/Amp (X-Gal) plates O/N.

1：
5

1：
3

1：
1

Blue
White

.5

1：
5

1：
3

1：
0

1：
1

60
40
200120
10080
60
40
200

1：
0

Relative colony number

120
10080

.5

Relative colony number*1High-fidelity PCR enzyme generates blunt ends.

Vector ： Insert （molar ratio）

Vector ： Insert （molar ratio）

DNA ： Ligation high Ver.2 = 1：1

DNA ： Ligation high Ver.2 = 1：0.5

Fig. 4 Blunt end ligation
Colony number on the condition of DNA : Ligation high Ver.2 = 1: 0.5 and Vector : Insert = 1 : 3
corresponds to 100.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

3NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio

Example 3.

Linker ligation

Relative colony number1201008060402012010080BlueWhite

604020DNA ： Ligation high Ver.2 = 1：1

Fig. 5

1：
101：
5.5
1：
01：
101：
51：
31：
1.5
1：
0Vector ： Insert （molar ratio）

1：
300

1：
1

Relative colony number

Dephosphorylated pUC19/HincII（50 ng, 25 fmol）was mixed at different ratios withphosphorylated HindIII linker (10mer). Half or an equal volume of Ligation high Ver.2,3.75 or 7.5 μL, was added and incubated at 16℃ for 30 min. E. coli DH5α competentcells were transformed using 10 μL of reaction mixture, and cultured on LB/Amp (X-Gal)
plates O/N.

Vector ： Insert （molar ratio）
DNA ： Ligation high Ver.2 = 1：0.5

Linker ligation

Colony number on the condition of DNA ： Ligation high Ver.2 = 1：0.5 and Vector ：Insert = 1：10
corresponds to 100.

Example 4.

TA cloning using purified PCR products

Relative colony number

T vector,（50 ng, 25 fmol）was mixed, at different ratios, with purified PCR products (500bp from Taq DNA polymerase). An equal volume of Ligation high Ver.2, 7.5 μL, was added
and incubated at 16℃ for 30 min. E. coli DH5α competent cells were transformed using
10 μL of reaction mixture, and cultured on LB/Amp (X-Gal) plates O/N.

120
100806040200BlueWhite

1： 0.5

1： 1

1： 3

1： 5Vector ： Insert （molar ratio）

Fig. 6 TA cloning using purified products
Colony number at 1:5 corresponds to 100

JAPAN
TOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

4NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio

Example 5.

TA Cloning using unpurified PCR products

Relative colony numberT vector,（50 ng, 25 fmol）was mixed, at different ratios, with unpurified PCR products(500 bp from Taq DNA polymerase). An equal volume of Ligation high Ver.2, 7.5 μL, wasadded and incubated at 16℃ for 30 min. E. coli DH5α competent cells were transformed
using 10 μL of reaction mixture, and cultured on LB/Amp (X-Gal) plates O/N.
120
1008060
40
200BlueWhite210.5

Amount of the added PCR products（μl）
Fig. 7TA cloning using unpurified products

Colony number on the condition of adding 0.5 μL of the PCR product corresponds to 100.

Example 6.

Phage vector ligation

λZAP®II (500 ng), EcoRI digested, and pRheo/EcoRI test insert（225 ng, 2.8 kb）, weremixed with half or with an equal volume of Ligation high Ver.2, 3.75 or 7.5 μL, andincubated at 16℃ for 30 min. in vitro packaging was performed with 1.5 μL ligation
reaction using GIGAPACK®III（Stratagene） followed by infection of E. coli XL-1 Blue
MRF’. The E. coli cells were cultured O/N and plaques counted.

Table 2.

Reaction conditions and efficiency
DNA soln：
Ligation reagent
Efficiency(pfu/μg ZAP®II)

1 ： 0.5

1 ： 1

6.3×106

5.5×106*λZAP® and GIGAPACK® are registered trademarks of STRATAGENE.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

5NOT FOR HUMAN OR DIAGNOSTIC USE.

www.toyobo.co.jp/e/bio[ 5 ] Troubleshooting
SymptomNo or low
colony number

Cause

Solution

Excess amount of ligation
mixture for transformation

Unpurified ligation mixtures should be added at less than10%(V/V) of competent cell suspensions.

Vector concentration is low

Increase the vector concentration.
(See [4 ] Application data)

DNA insert fragment
concentration is too low.

Increase the DNA insert fragment concentration.
(See [4 ] Application data)

Excess salts

・DNA fragments should be dissolved in a low salt solution.
・Unpurified PCR products should be added at 0.5-1 μL perligation reaction.
Dephosphorylated vectors cannot ligate with unphosphorylated
PCR products.

Ends of both vector andinsert are dephosphorylated.
Cloning efficiency waslow

Primer dimer

When the PCR reaction contains primer dimers or unexpected
PCR products, the desired PCR product should be purified.

Insufficient time for ligation
reaction.
Insert fragment
concentration is too low.

The reaction can be prolonged for up to 2hr.

Increase the insert fragment concentration.
(See [4 ] Application data)

Self-ligation ofvector

Use dephosphorylated vector.

Excess number ofcolonies

Self-ligation ofvector

Use dephosphorylated vector.

Freezing of the reagent

Storage conditions

In the case of -30℃ storage or long-term storage under -20℃,
the reagent might freeze. The frozen reagent can be thawed byincubation at room temperature in short time. No decrease inreaction efficiency is observed following 5 freeze-thaw cycles.

White precipitates areformed

Storage condition

In the case of -30℃ storage or long-term storage under -20℃,
the reagent might form white precipitates. The precipitatesshould be dissolved prior to use. The precipitates can bedissolved by incubation at room temperature with vortexing atlow speed in short time.

JAPANTOYOBO CO., LTD.
Tel(81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY.

6NOT FOR HUMAN OR DIAGNOSTIC USE.