TA-Blunt Ligation Kit Manual （3rd edition）
Code No. 315-06541

For 5 reactions

Code No. 311-06543

For 50 reactions- Description Nippon Gene has been offering the “Ligation-Convenience Kit” which can be used to perform DNA ligation
reactions simply and efficiently, in contrast to the long reaction times of the conventional ligation method
using T4 DNA Ligase singly. However, in general, ligation efficiency varies depending on the shape ofthe DNA ends.
“TA-Blunt Ligation Kit” is a ligation kit specialized for TA cloning and blunt end ligation using a buffer ofNippon Gene’s own composition. “TA-Blunt Ligation Kit” offers higher DNA ligation efficiency than the“Ligation-Convenience Kit”.

I. Characteristics● This product is a kit for performing highly efficient TA cloning and ligation of blunt end DNA.
● The 5×Ligation Mix includes a reaction buffer required for DNA ligation, ATP, DTT, T4 DNA Ligase andthe like. Highly efficient DNA ligation reactions can be performed by adding 1/5 of the volume of the 5×Ligation Mix and 1/10 of the volume of the 10×Enhancer solution to the reaction system.
● Since the 5×Ligation Mix and the 10×Enhancer solution are not frozen at -20°C, these can be used
immediately after taking out of the refrigerator and do not require complicated thawing operations.
● After completing the ligation reaction, the product can be used for transformation as-is.

II. Contents of kit
Reagent

For 5 reactions* For 50 reactions*

5×Ligation Mix

20 μl×1

100 μl×2

10×Enhancer solution

10 μl×150 μl×2

* Number of reactions when used in 20 μl reaction system
Vector DNA is not included in this kit.

III. Storage method－20°C storage・ Since this product is not frozen at -20°C storage, there is no need to worry about reduction of stability
due to freezing and thawing, and there is no need for complicated thawing operations.
・ Handle on ice during usage.

-1-IV. Protocol and experimental examples
1. Protocol(1) Preparation of DNA solution

Vector DNA

Prepare 14 μl of DNA solution by combining a proper
molar ratio of vector DNA (about 0.025 pmol) andinsert DNA fragment.*1(2) Preparation of ligation reaction solution
Add 2 μl of 10×Enhancer solution to 4 μl of 5×Ligation Mix and mix.*2

Insert DNAup to 14 μl

ddH2O or TE *1
10×Enhancer solution

2 μl

5×Ligation Mix

4 μlTotal20 μl↓
React at 16°C for 30 min.*3(3) Ligation reaction↓React at 16°C for 30 min.*3

Transformation*4-6(4) Transformation
Use the reaction solution for transformation as-is.*4-6* The ratio of the amount of vector to 10×Enhancer solution is very important.
Use about 0.025 pmol vector for the reaction in 20 μl reaction system（about 50 ng in the case of
3 kbp vector）.
* When the ligation reaction is performed in a 10 μl reaction system, use half the amount of DNA,
10×Enhancer solution and 5×Ligation Mix that would be used in a 20 μl reaction system.

2. Investigation of the vector : insert molar ratio
The vector : insert molar ratio in ligation greatly affects ligation efficiency. The tables below show themolar ratios at which the best results were obtained at Nippon Gene when insert DNAs of various lengths
were ligated and used for transformation under the conditions described below.*3
1. Plasmid vector ligation(1) Blunt end ligation
Insert sizeVector
Insert200 bp 500 bp16-10

13-6

1 kbp

3 kbp

1
3

1
0.5-1

(2) TA cloning
Insert size
VectorInsert200 bp 500 bp

1 kbp

3 kbp

16-10

11-6

1
0.5-1

13-6

Vector： pBluescript II SK(+) cleaved with EcoR V.
Stratagene Inc. (0.025 pmol)
Insert： Insert DNA cleaved with EcoR V(0.0125 pmol, 0.025 pmol, 0.0375 pmol, 0.15 pmol, 0.25 pmol)
Ligation reaction： 16°C, for 30 min.

Vector： pGEMⓇ-T
Promega Inc. (0.025 pmol)
Insert： PCR productamplified by Gene Taq NT (Code No. 318-03231)(0.0125 pmol, 0.025 pmol, 0.0375 pmol, 0.15 pmol, 0.25 pmol)
Ligation reaction： 16°C, 30 min

2. Linker ligation
Insert size

8 bp

VectorInsert1700-1000

Vector： pUC19 (0.03 pmol) cleaved by Hinc II
Insert： Linker EcoR V(0.09 pmol, 0.3 pmol, 1.5 pmol, 3 pmol, 6 pmol, 15 pmol, 21 pmol, 30 pmol)
Ligation reaction： 16°C, 30 min-2-3. Experimental example< Experimental example 1: TA cloning >Comparison to T4 DNA Ligase(1)

A 500 bp DNA fragment derived from lambda DNA was PCR amplified with Taq DNA Polymerase.

(2)

Fourteen μl of DNA solution containing 50 ng of pGEMⓇ-T vector (0.025 pmol) and 25 ng of insert
DNA fragment (insert/vector molar ratio is about 3) was prepared.*1(3)

Two μl of 10×Enhancer solution and 4 μl of 5×Ligation Mix were added to 14 μl of the DNA solution
and mixed.

(4)

The mixture was reacted at 16°C for 5 min to 16 hrs.

(5)

After the reaction, 1 μl of the reaction solution was used for transforming 30 μl of JM109 competentcells, and the generated colonies were counted. Also, as a control, a similar ligation reaction wasperformed using T4 DNA Ligase as a single body.

The transformation efficiency of JM109

8

competent cells used in this experiment was 1.41×10 cfu/μg（pBR322 DNA）.*4, 5
Effect of reaction time in TA cloning
400

100

350

90

White colony
Blue colony

30080

White colony rate(%)

250

60

200

50
40

150

30
1002050

White colony rate (%)

Colony number

70

1000
16 hr

3 hr

1 hr

30 min

5 min

16 hr

3 hr

1 hr

30 min5 min

T4 DNA Ligase

TA-Blunt Ligation Kit

Ligation reaction time and obtained number of white colonies
Reaction time

5 min30 min

1 hr

3 hrs

16 hrsTA-Blunt Ligation Kit
T4 DNA Ligase

99120721540176
0171
7Result
When the TA-Blunt Ligation Kit was used, it was found that ligation was sufficiently completed within areaction time of 30 min.

-3-< Experimental example 2: blunt end ligation >Comparison with T4 DNA Ligase(1)

pBluescript II SK(+) was cleaved with EcoR V, dephosphorylated, extracted withphenol/chloroform/isoamyl alcohol, precipitated with ethanol and then dissolved in TE buffer.*1(2)

A 1000 bp DNA fragment derived from lambda DNA was cleaved with EcoR V.

(3)Fourteen μl of DNA solution containing 50 ng of pBluescript II SK(+) and 50 ng of insert DNA
fragment (insert/vector molar ratio is about 3) were prepared.*1(4)

Two μl of 10×Enhancer solution and 4 μl of 5×Ligation Mix were added to 14 μl of DNA solution andmixed.

(5)

The mixture was reacted at 16°C for 5 min to 16 hrs.

(6)

After the reaction, 1 μl of the reaction solution was used for transforming 30 μl of JM109 competentcells, and the generated colonies were counted. Also, as a control, a similar ligation reaction wasperformed using T4 DNA Ligase as a single body.

The transformation efficiency of JM109

8competent cells used in this experiment was 1.41×10 cfu/μg (pBR322 DNA).*4, 5

Effect of reaction time on blunt end ligation
200

Blue colony

100180White colony rate(%)

90

160

80

14070

120

60

100

5080

4060

30

40

2020

1000
16 hr

3 hr

1 hr

30 min

5 min

16 hr

3 hr

1 hr

30 min5 min

T4 DNA LigaseWhite colony rate (%)

Colony numberWhite colony

TA-Blunt Ligation Kit

Ligation reaction time and obtained number of white colonies
Reaction time

5 min

30 min

1 hr

3 hrs

16 hrsTA-Blunt Ligation Kit
T4 DNA Ligase

129
5143
22116
16155
50

129
102

ResultWhen the TA-Blunt Ligation Kit was used, it was found that ligation was sufficiently completed within areaction time of 30 min.

-4-< Experimental example 3: ligation reaction at a room temperature of 25ºC >Comparison of 16ºC and 25ºC
（1） pBluescript

IISK(+)

wascleavedwithEcoRV,dephosphorylated,extracted

with*1

phenol/chloroform/isoamyl alcohol, precipitated with ethanol and then dissolved in TE buffer.
（2） A 1000 bp DNA fragment derived from lambda DNA was cleaved with EcoR V.

（3） Fourteen μl of DNA solution containing 50 ng of pBluescript II SK(+) and 50 ng of insert DNAfragment (insert/vector molar ratio is about 3) were prepared.*1（4） Two μl of 10×Enhancer solution and 4 μl of 5×Ligation Mix were added to 14 μl of DNA solution andmixed.
（5）

The mixture was reacted at 25ºC or 16°C for 5 min to 30 min.

（6） After the reaction, 1 μl of the reaction solution was used for transforming 30 μl of JM109 competentcells, and the generated colonies were counted. Also, as a control, a similar ligation reaction wasperformed using T4 DNA Ligase as a single body.

The transformation efficiency of JM109

competent cells used in this experiment was 1.41×108 cfu/μg （pBR322 DNA）.*4, 5Comparison of ligation efficiency at 16°C and 25°C
800

10090700

80600
500604005040300

White colony rate (%)

Colony number70302002010010White colony
Blue colony0016℃ 5 min

25℃ 5 min

16℃ 30 min

25℃ 30 min

White colony rate （％）

ResultLigation could be performed at 25°C with a ligation efficiency equivalent to 16°C.

-5-V. Caution*1

Use about 0.025 pmol of the vector in the ligation reaction.
Also, when DNA is dissolved in a buffer containing a high concentration of salts and a buffer
containing a high concentration of EDTA, ligation efficiency is markedly reduced. Prepare DNA
solution in ddH2O or TE buffer (pH 8.0).

*2

Measure the amount of 10×Enhancer solution to be added precisely. If more than or less than adesignated amount is added, ligation efficiency may be markedly reduced.

*3

Ligation efficiency may vary depending on the purity of DNA used for the ligation reaction or therestriction enzyme employed.

*4

The volume of the reaction solution used for transformation should be not more than 1/10 of thevolume of the competent cells. If too much reaction solution is used, transformation efficiency may
be reduced.

*5

If the volume of the reaction solution is 1/10 of the volume of the competent cells or more, treat thereaction solution with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol(24:1) after the ligation, recover DNA by ethanol precipitation, dissolve DNA in ddH2O or TE buffer(pH 8.0) so that the volume is not more than 1/10 of the volume of the competent cells, and thenperform transformation.

*6

When the transformation is performed by the electroporation method, treat the ligation product withphenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1), recover DNA byethanol precipitation, dissolve DNA in ddH2O or TE buffer (pH 8.0), and then perform transformation.

Q＆A
Q: How should the ligation product be stored?
A: Store the product at -20°C. The product can be used for transformation experiments as-is afterthawing.
Q: We have many samples. Would it be possible to prepare a pre-mix by mixing 10×Enhancer solution
and 5×Ligation Mix before use?
A: You may prepare the pre-mix before use. However, prepare the pre-mix immediately before use anduse it up immediately after preparing.
Q: Can the ligation reaction be performed at 4°C overnight?
A: Yes, the ligation reaction can be performed at 4°C overnight (16 hrs).
Q: Can the ligation product be heat-treated?
A: Yes, the ligation product can be treated at 70°C for 10 min.

-6-Q: Can the ligation product be ethanol precipitated as-is?
A: When the ligation product is ethanol precipitated as-is, the number of colonies are markedly reduced.
Perform ethanol precipitation after purifying with phenol/chloroform according to the method describedin the manual(*5).

Also, DNA can be recovered efficiently by ethanol precipitation using

Ethachinmate.
Q: Can the ligation reaction be performed using DNA prepared by ethanol precipitation using
Ethachinmate?
A: Yes.

VI. Troubleshooting
TroubleProbable cause

Countermeasure

Mismatch of ends

Check ends.

Treat the ligation product withNo ligation(no colony formation)

The ligation product is not purified phenol/chloroform/isoamyl alcohol
when transformation is performed by(25:24:1) and chloroform/isoamyl
electroporation.
The ligation productprecipitated as-is.

isalcohol (24:1), recover DNA byethanol precipitation, dissolve DNA inethanol ddH O or TE buffer (pH 8.0), and2
then perform transformation.

Adjust the amount of the vector toabout 0.025 pmol in the 20 μl reaction
system.
Adjust Enhancer solution to be added
There is too little or too much Enhancer
to 1/10 of the volume of the reaction
solution.
system.
Add Enhancer solution at 1/10 of theEnhancer solution is not added.
volume of the reaction system.
Dissolve DNA in TE buffer or water not
Concentration of the chelating agent
containing the chelating agent andsuch as EDTA is too high.
perform the ligation.
Dissolve DNA in TE buffer or water not
Salt concentration of the DNA solution
containing salts and perform theis too high.
ligation.
There is too little or too much vector.

Low ligation efficiency

Ligation time is too short.

Extend the ligation time.

The insert:vector ratio is not suitable.

Perform the ligation at an optimum
molarratio
by consulting
theinsert:vector ratio in the manual.

The vector has sticky ends.

Low transformation
efficiency

This product cannot be used for sticky
end ligation.

We recommend to use

Ligation-Convenience Kit.
Adjust the reaction solution to be used
for the transformation to 1/10 of thevolume of the competent cells.
TMWhen ECOS
Competent E.coli is
Too much reaction solution is used.
used, the volume of the reaction
solution to be added should be kept at
5% or lower to the competent cells.
Use competent cells having theLow transformation efficiency of thetransformation efficiency of not lesscompetent cells.
7than 1ｘ10 cfu/μg.

-7-High ratiocoloniesofblue

Dephosphorylation of the blunt end Confirm the presence or absence ofvector is insufficient.
self-ligation beforehand.
The T protrusion of the T vector is Use the T vector in which the Tmissing.
protrusion is preserved.

NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel+81-76-451-6548
URL https://www.nippongene.com/TA-Blunt Ligation Kit Manual (ver.3) 151209HT-8-