Ligation-Convenience Kit Manual (4th edition)

Code No. 319-05961

For 100 ligations

Code No. 315-05963

For 10 ligations- Description –The first important step of gene cloning is to ligate the target gene to a vector DNA. The efficiency of theligation reaction varies depending on the shape of the DNA terminal, and until now the ligation reaction
has needed to be optimized for each reaction. Further, the ligation reaction requires 1-16 hours using a
conventional T4 DNA ligase, and it has been difficult to obtain high transformation efficiency.

The

Ligation-Convenience Kit can solve these problems and provide highly efficient ligation in a short periodof time.

I. Characteristics● This product is a kit for performing DNA ligation rapidly and simply.
● The 2×Ligation Mix contains all the components required for DNA ligation such as the reaction buffer,ATP, DTT, T4 DNA Ligase, etc. The DNA ligation reaction can be performed by adding an equalvolume of 2×Ligation Mix to the DNA solution.
● By using this kit, the ligation reaction can be performed in 5-30 min regardless of the shape of theDNA terminal. Further, the DNA solution in which the ligation reaction has been completed can beused for transformation and in vitro packaging as-is.

II. Contents of kit
Reagent

For 100 ligations *For 10 ligations *2×Ligation Mix

250 μl×4

100 μl×1* When used in 20 μl reaction system

III. Storage and melting method
Storage at －20℃
・ At the time of use, thaw completely on ice and mix well by pipetting.
・ No reduction in reaction efficiency was found in up to 50 cycles of freezing and thawing.

Efficiency

Stability against freeze and thaw
1.4
1.2
10.8
0.6
0.4
0.2
005

1020 30 40cycles

50

Frequency of freeze and thaw-1-IV. Protocol and examples of experiments
1. Protocol(1) Preparation of DNA solution
Prepare 10 μl of DNA solution by mixing avector DNA and an insert DNA fragment at
a proper molar ratio.*2

Vector DNAInsert DNA
ddH2O or TE(2) Preparation of ligation reaction
solution
Add an equal volume of 2×Ligation Mix tothe DNA solution and mix.

up to 10 μl*22×Ligation Mix

10 μl

Total

20 μl

React at 16°C for 5-30 min.*1(3) Ligation reaction

React at 16°C for 5-30 min.*1(4) Transformation or in vitro packaging

Transformation or in vitro
packaging *4-9

Use the reaction solution for transformation
or in vitro packaging as-is.*4-9
2. Investigation of the vector : insert molar ratio
The vector : insert molar ratio in ligation has a significant effect on the ligation efficiency. Shown below
are the results obtained at Nippon Gene when ligating various length of inserts and transforming withthem. The molar ratios which gave the best results under the specific conditions are summarized in thetable below.＊3
1. Plasmid vector ligation(1) Sticky end
Insert length

200 bp600 bp1000 bp3000 bp

VectorInsert

110

15-10

12-10

10.5-2Insert length

200 bp600 bp1000 bp3000 bp

VectorInsert

1515

12-10

10.5-2Insert length

200 bp600 bp1000 bp3000 bp

VectorInsert

110

15

15

11

Vector: pUC19 (0.03 pmol) cut by EcoR I
Insert: insert DNA (0.015 pmol, 0.03 pmol, 0.06 pmol,0.15 pmol, 0.3 pmol) cut by EcoR I
Ligation reaction: 16°C, 5 min(2) Blunt end
Vector: pUC19 (0.03 pmol) cut by Sma I
Insert: insert DNA (0.015 pmol, 0.03 pmol, 0.06 pmol,0.15 pmol, 0.3 pmol) cut by Sma I
Ligation reaction: 16°C, 5 min(3) TA cloning
Vector: pGEMⓇ-T Easy; Promega Inc. (0.03 pmol)
Insert: PCR product amplified by Gene Taq NT (Code No.
318-03231)(0.015 pmol, 0.03 pmol, 0.06 pmol,0.15 pmol, 0.3 pmol)
Ligation reaction: 16°C, 5 min

2. Linker ligation

Insert length
VectorInsert8 bp1Not less than 50

Vector: pUC19 (0.03 pmol) cut by Hinc II
Insert: Linker EcoRV (Code No. 318-02251)(0.3 pmol , 1.5 pmol , 3 pmol , 15 pmol)
Ligation reaction: 16°C, 5 min-2-3. Examples of experiments< Example (1) Sticky end ligation >Comparison with T4 DNA Ligase
(1)

pUC19

DNA

wascutwith

EcoRI,dephosphorylated

andextracted

withphenol/chloroform/isoamyl alcohol, and then dissolved in TE buffer.*2(2)

A 500 bp DNA fragment derived from lambda DNA was cut with EcoR I.

(3)

10 μl of DNA solution containing 50 ng of pUC19 DNA and 20 ng of the insert DNA fragment(insert/vector molar ratio≒2) was prepared.*2(4)

10 μl of 2×Ligation Mix was added to 10 μl of DNA solution and mixed.

(5)

The reaction was carried out at 16°C for 5 min to 16 hｒ.

(6)After the reaction, 50 μl of competent cells of JM109 was transformed with 5 μl of the reaction
solution, and the number of developed colonies was counted. Also, as a control, a similar
ligation reaction was performed using T4 DNA Ligase by itself． Further, the transformation
efficiency of the competent cells of JM109 is 1.3×108 cfu/μg (pBR322 DNA).*4, 510090807060504030
20100

Colony number

600
500
4003002001000W hite c ol ony

16 hr

1 hr

30 min

5 min

16 hr

1 hr

30 min5 min

T4 DNA Ligase

White colony ratio （%）

Effect of reaction time in sticky end ligation
700

Blue colony
W hite c ol ony ratio(%)

Ligation-Convenience Kit

Relation between ligation reaction time and transformation efficiency (white colony count)
Reaction time

5 min

30 min

1 hr

16 hr

Ligation-Convenience Kit

450390323

7T4 DNA Ligase

145

123

14623[ Results ]The above results indicate that the ligation reaction is sufficiently completed with a reaction time of 5 minwhen the Ligation-Convenience Kit is used. When the ligation time is extended to 16 hr, however, thetransformation efficiency tends to decrease.*3-3-< Example (2) Blunt end ligation >Comparison with T4 DNA Ligase(1)

pBluescript

IISK(+)

wascutwith

EcoRV,dephosphorylated

andextracted

withphenol/chloroform/isoamylalcohol, and then dissolved in TE buffer.

*2

(2)

A 500 bp DNA fragment derived from lambda DNA was cut with EcoRV.

(3)

10 μl of DNA solution containing 50 ng of pBluescriptⅡSK(+) and 20 ng of the insert DNA
fragment (insert/vector molar ratio≒2.4) was prepared.*2(4)

10 μl of 2×Ligation Mix was added to 10 μl of the DNA solution and mixed.

(5)

The reaction was carried out at 16°C for 5 min to 16 hr.

(6)After the reaction, 50 μl of competent cells of JM109 was transformed with 5 μl of the reaction
solution, and the number of developed colonies was counted.

Also, as a control, a similar

ligation reaction was performed using T4 DNA Ligase by itself．

Further, the transformation

efficiency of the competent cells of JM109 is 1.3×108 cfu/μg (pBR322 DNA).*4, 5

Effect of reaction time in blunt end ligation

Colony number

12080

100608060

40402020

White colony ratio (%)

100

14000
16 hr

60 min30 min

5 min

16 hr

60 min30 min5 min

T4 DNA Ligase

White colony
Blue c olony
White colony rat io(% )Ligation-Convenience Kit

Ligation reaction time and obtained white colony count (number of white colonies)

Reaction time

5 min

30 min

1 hr

16 hr

Ligation-Convenience Kit

615957

0T4 DNA Ligase912111[ Results ]It is found that the ligation reaction is sufficiently completed with a reaction time of 5 min when theLigation-Convenience Kit is used.

When the ligation time is extended to 16 hr, however, thetransformation efficiency tends to decrease.*3-4-

< Example (3) Linker ligation >Comparison with T4 DNA Ligase (investigation of linker : vector molar ratio)(1)

pUC19 DNA was cut with Hinc II, dephosphorylated and extracted with phenol/chloroform/isoamyl
alcohol, and then dissolved in TE buffer.*2(2)Each 10 μl DNA solution was prepared by mixing 50 ng of pUC19 DNA vector (about 0.03 pmol)
with linker DNA so that the molar ratio (linker : vector) was 10, 50, 100 or 500.*2(3)

10 μl of 2×Ligation Mix was added to each 10 μl of DNA solution and mixed.

(4)

The reaction was carried out at 16°C for 5 min.*1(5)

After the reaction, 50 μl of competent cells of JM109 was transformed with 5 μl of the reaction
solution, and the number of developed colonies was counted.

Also, as a control, a similar

ligation reaction was performed using T4 DNA Ligase by itself． The transformation efficiency ofthe competent cells of JM109 was 1.3×108 cfu/μg (pBR322 DNA).*4, 550001004000803000602000401000

20White colony ratio (%)

Colony number

Effect of linker/vector molar ratio on linker ligation

W hite c olony
Blue colony

00500

10050

10500

10050

10T4 DNA Ligase

W hite c olony ratio(%)(Linker／Vector molar ratio）

Ligation-Convenience Kit[ Results ]The results above indicate that linker ligation efficiency is higher when the linker : vector molar ratio is50-100.*3< Example (4) Ligation to cosmid vector and in vitro packaging >(1)

10 μl mixture containing 1 μg of Charomid 9-36 (Code No. 311-01401) linearized with EcoR I and0.5 μg of a 4 kbp DNA fragment derived from ColE1 (insert : vector molar ratio≒4.5) wasprepared.*2(2)

10 μl of 2×Ligation Mix was added to 10 μl of the DNA solution and mixed.

(3)

The reaction was carried out at 16°C for 5 min to 16 hr.

(4)Using 3 μl of the reaction-completed solution (1/10 volume of the packaging Extract) andLAMBDA INN (Code No. 317-01741), in vitro packaging operation was carried out.*6～8-5-(5)Also, as a control, the ligation reaction using T4 DNA Ligase was carried out and a similar in vitro
packaging was carried out.

60

Effect of ligation time on in vitro

Colony number

50
40
30
20
10Ligat ion-Convenienc e Kit
T4 DNA Ligas e

05 min30 min1 hr
Reaction time

16 hr

[ Results ]When the Ligation-Convenience Kit is used, it is observed that a ligation reaction time of 5-30 min issufficient. Conversely, the packaging efficiency became worse by prolonging reaction time.

When T4

DNA Ligase was used by itself, the packaging efficiency did not improve much by reacting for 16 hr.*3

V. Notes*1

When ligation is carried out over a long duration, e.g., 16 hr, overnight or the like,transformation efficiency may be markedly reduced.

*2

When DNA is dissolved in a high salt concentration buffer, ligation efficiency is markedly reduced.
Prepare the DNA solution in ddH2O or TE buffer (pH8.0).

*3

Ligation efficiency may be different depending on the purity of DNA used for the ligation reaction andon the employed restriction enzyme.

*4

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

*5

If the volume of the reaction solution is 1/10 of the competent cells by volume or more, recover theDNA after the ligation reaction by ethanol precipitation, dissolve the DNA in ddH2O or TE buffer(pH8.0) so that the volume of the DNA is not more than 1/10 of the competent cells by volume, andthen carry out the transformation.

*6

The volume of the reaction solution to be used for packaging should be not more than 1/10 of thepackaging Extract by volume. If too much of the reaction solution is used, the packaging efficiency
may be reduced.

*7

If the volume of the reaction solution is 1/10 of the packaging Extract by volume or more, recover theDNA after the ligation reaction by ethanol precipitation, dissolve the DNA in ddH2O or TE buffer(pH8.0) so that the volume of the DNA is not more than 1/10 of the packaging Extract by volume,and then carry out the transformation.
-6-*8

Packaging is not inhibited by the use of Gigapack（Stratagene Inc.）.

*9

Heat treatment of the ligation reaction completed solution causes a marked reduction oftransformation efficiency.

When the heat treatment is performed, heat the ligation reaction

completed solution at 70°C for 10 min after diluting the ligation reaction completed solution two foldwith ddH2O (DNase-free).

VI. Troubleshooting
Problem
No ligation

Low ligation efficiency

Low transformation efficiency

Presumed cause

Countermeasure

Mismatch of the ends

Identify the ends

Concentration of 2×Ligation Mixafter thawing is nothomogeneousMix well by pipetting afterthawing on ice.

Contamination of short DNA
fragments

Remove the short DNA
fragments generated byrestriction enzyme reaction byelectrophoresis or the likeHigh salt concentration

Dissolve DNA in water or TE
buffer containing no salts, andperform ligation.

Long ligation time

Perform ligation within 1 hr.

Use of too much reaction solution

Limit the reaction solution used
for transformation within 1/10
volume of competent cells.

Insufficient vector
dephosphorylation

Determine beforehand whether
there is self-ligation or not.

Heat treatment without diluting
the ligation reaction solution after
completing the reaction

After completing the reaction,
dilute the ligation reaction
solution twofold with ddH2O(DNase-free) and then perform
heat treatment (70°C for 10 min).

Low transformation efficiency ofthe competent cells

Use competent cells having atransformation efficiency of 5 x
107 cfu/μg (pBR322 DNA) orhigher.

-7-NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel076-451-6548
URL https://www.nippongene.com/

Ligation-Convenience Kit Manual (ver.4) 120105KH-8-