Blunting-Convenience Kit Manual （2nd edition）

Code No. 312-06291

For 25 reactions

Code No. 318-06293

For 5 reactions- Description When no appropriate restriction enzyme is available for linking the target gene to vector DNA, ligation
can be performed without depending on sequence if the DNA ends are blunt. T4 DNA Polymerase is aDNA-dependent DNA polymerase having 3’→5’ Exonuclease activity and 5’→3’ Polymerase activity,and both 5’ protruding end and 3’ protruding end can be blunted with this enzyme. Also, by combining
with the 2×Ligation Mix (Ligation-Convenience Kit) by which the ligation reaction can be performed in ashort period of time, the reactions from blunting to ligation can be performed in a short period of time.

I. Characteristics● Reactions from blunting DNA ends to ligation can be performed rapidly and simply.
● Since the 10×Blunting Buffer includes dNTP and the like required for the reaction, the blunting ofDNA ends can be performed simply by adding T4 DNA Polymerase.
● 5’ protruding end DNA, 3’ protruding end DNA and PCR products with A at 3’ ends can be blunted.
● Ligation reaction can be performed in a short period of time using the 2×Ligation Mix.

II. Contents of kit
Reagent

For 25 reactions*

For 5 reactions*

T4 DNA Polymerase

25 μl×15 μl×1

10×Blunting Buffer

50 μl×110 μl×1

2×Ligation Mix

250 μl×150 μl×1

* Number of reactions when used in 20 μl reaction system for both blunting and ligation.

III. Storage and thawing method
Store at −20°C
・ To avoid denaturation, never vortex T4 DNA Polymerase.
・ Use 2×Ligation Mix after completely thawing on ice and mixing well by pipetting.
・ No decrease of ligation efficiency of 2×Ligation Mix was observed in up to fifty cycles of freezing
and thawing.

-1-IV. Protocol and examples of experiments< 1. Protocol >(1) Preparation of blunting reaction solution< Blunting reaction >Prepare 17 μl solutions of DNA to be blunted such as 5’

Sample DNA solution (0.1-10 pmol) 17 μl

protruding end DNA, 3’ protruding end DNA and PCR

10×Blunting Bufferproducts (at 0.1-10 pmol DNA end concentration).

＊1

Then add 2 μl of 10×Blunting Buffer and mix.

2 μl

Add 1 μl of T4 DNA Polymerase(2) Addition of enzyme
Add 1 μl of T4 DNA Polymerase and mix by pipetting.＊2

React at 37ºC for 5 min.

(3) Blunting reaction
React at 37°C for 5 min.＊3

Stop the reaction by placing the mixture on(4) Stopping the reaction

ice. (Blunting reaction solution)

Place the blunting reaction mixture on ice to stop thereaction.

When not used immediately, performphenol/chloroform/isoamyl

alcohol

extraction* Use the required
amount of blunting
reaction solution.
6（1-5 μl）＊andethanol precipitation and store the sample at -20°C.
(5) Preparation of ligation reaction solution
Prepare 10 μl of DNA solution by combining the< Ligation reaction >Blunting reaction solution

blunting reaction solution and the blunt end DNA vector

Blunt end DNA vector solution up to 10 μlsolution.

ddH2O

Add 10 μl of 2×Ligation Mix and mix.

2×Ligation Mix

10 μl

Total

20 μl

(6) Ligation reaction
React at 16ºC for 5-30 min.＊4(7) Transformation

React at 16ºC for 5-30 min.

Use the reaction solution for transformation as it is.＊5
For transformation or in vitro packaging*1 DNA end concentration of 0.1-10 pmol is equivalent of 0.1-10 μg of pUC19 (2686 bp).
*2 Never vortex T4 DNA Polymerase. The enzyme may be inactivated.
*3 Strictly observe the reaction time of 5 min.
*4 When ligation is carried out for a long time, such as 16 hours or overnight,
transformation efficiency may be markedly decreased.
*5 The volume of the reaction solution used for transformation should be not more than
1/10 of the volume of the competent cells. If too much reaction solution is used,transformation efficiency may be decreased.
If the volume of the reaction solution is not less than 1/10 of the volume of thecompetent cells, recover the DNA after the ligation reaction by ethanol precipitation,
dissolve the DNA in ddH2O or TE (pH 8.0) so that the volume of the DNA is not morethan 1/10 of the volume of the competent cells, and then use for transformation.
*6 The blunting reaction solution brought into the ligation reaction should be not more than-2-

5 μl.

If the volume of the reaction solution is not less than 5 μl, recover the DNA by

extracting with phenol/chloroform/isoamyl alcohol and precipitating with ethanol,
dissolve in ddH2O or TE (pH 8.0) and then use the DNA.

< 2. Experimental example >Confirmation of blunting of the vector
1) pUC19 was cleaved with EcoR I (5’ protruding end) and Pst I (3’ protruding end),
extracted with phenol/chloroform/isoamyl alcohol and then dissolved in TE buffer.
2) The ends of 1 μg of pUC19 cleaved with EcoR I and Pst I were blunted in accordancewith the protocol.
3) A portion of the blunted DNA was subjected to the ligation reaction at 16°C for 30 min
using 2×Ligation Mix.
4) After the ligation reaction, to remove uncut pUC19 and to reduce the background, theligation reaction solution was diluted five-fold and heated at 65°C for 5 min to inactivate
T4 DNA Ligase, and was digested with BamH I.
5) With the ligation reaction solution digested with BamH I, ECOSTM Competent E.coli
DH5α was transformed and the number of colonies generated was counted.
The ratio of the number of colonies generated by self-ligation after blunting is shown
below when the number of colonies generated by the transformation by pUC19 isassumed to be 1.
ベクターの平滑化の確認
Confirmationof vector blunting

Result1.20

Blue colony
White colony1.00

When pUC19 is digested with EcoR I
and Pst I, blunted and self-ligated, theLacZ frame is in phase generating bluecolonies. Since blue colonies derived

0.80

from pUC19 are excluded by digestingwith BamH I, the generated blue

0.60

colonies are the plasmid formed by
self-ligation after blunting the ends.
0.40

The left graph indicates that about 60%of the vector is blunt-end ligated after 5
min reaction.

0.20

0.00

pUC19
pUC19

Blunted
平滑化あり

Not
blunted
平滑化なし-3-

Blunting of insert DNA1)

pBluescript II SK(+) was cleaved with EcoR V and dephosphorylated, thenextracted with phenol/chloroform/isoamyl alcohol and then dissolved in TE.

2)

Two kinds of fragments were prepared by cleaving the 500 bp fragment, which wasamplified using λDNA as a template, with EcoR I and Pst I.

3)

Two fragments were blunted in accordance with the protocol.

4)

The vector, which was obtained by cleaving pBluescript II SK(+) with EcoR V and bydephosphorylating, and the blunted fragment were ligated at an insert/vector molar
ratio of 5 at 16°C for 30 min using 2×Ligation Mix.

Also, the fragment, which wasamplified as a control using λDNA as a template, was cleaved with EcoR V andligated.
ECOSTM Competent E.coli DH5α was transformed with a portion of the ligation5)

reaction solution, and the number of generated colonies was counted.
Blue colony
White colony

Eco
RI I インサート
EcoR
Insert

80.0
70.0

800

60.0
50.0

600

40.0
400

30.0
20.0

200

10.00

Not平滑化なし
blunted

90.0

600

80.0
500

70.0

400

60.0
50.0

300

40.0

200

30.0
20.0

100

10.000.0
平滑化あり
Blunted

100.00.0

Blunted平滑化あり

Control
Control（EcoRⅤfragment）（EcoRV fragment）

White白コロニー率
colony ratio (%)

コロニー数
Colonynumber1000

コロニー数
Colonynumber90.0

White colony rate (%)

700

100.0

White白コロニー率
colony ratio (%)

1200

PstIIインサート
Pst
Insert

平滑化なし
Not
bluntedControl
Control（EcoRⅤVfragment）（EcoRfragment）

ResultFor both 5’ end protrusion and 3’ end protrusion, the blunted insert could be ligated at thesame ratio as the EcoR V fragment, suggesting that the EcoR I fragment and Pst I fragment
were sufficiently blunted by the reaction at 37°C for 5 min.

-4-< 3. Reference data >1. Blunting PCR products
A 500 bp fragment amplified by PCR was blunted in accordance with the protocol.
(1) One portion was ligated as-is after blunting to pBluescript II SK(+) cleaved withEcoR V.
(2) The other portion was phosphorylated with T4 Polynucleotide Kinase after
blunting, treated with heat to inactivate T4 Polynucleotide Kinase, and then ligatedwith pBluescript II SK(+), which was cleaved with EcoR V and dephosphorylated.*
The graph below shows the ratio of blue colonies to white colonies when the number ofcolonies generated is assumed to be 1.
Presence or absence of phosphorylation after blunting
平滑化後のリン酸化の有無について

ResultWhen a PCR product was blunted and ligatedas-is with a vector having a phosphate group, theBlue colony

1.20

White colony

ratio of white colonies was about 13%. On theother hand, when the PCR product was

1.00

phosphorylated
0.80

andligatedwiththedephosphorylated vector, the ratio of whitecolonies was not less than 90%.

0.60

When a PCR product is used for ligation after
0.40

blunting, the phosphate group is normally not
attached to the 5’ end of the PCR product and0.20

thus a vector that is not dephosphorylated needs0.00
リン酸化無し①

リン酸化あり②to be used.

However, when the vector that isnot dephosphorylated is used for ligation, theratio of the self-ligated vector is greater.
Therefore, when the ligation is performed byblunting the PCR product, it is recommended
that the PCR product be phosphorylated using
T4 Polynucleotide Kinase after blunting and tothat the vector be dephosphorylated.

* When T4 Polynucleotide Kinase (Code No. 312-01551) from Nippon Gene is used, andphosphorylation is performed using 10×Kinase Buffer A, the ligation may be performed
using the 2×Ligation Mix after heat treatment without further processing.
When phosphorylation is performed using 10×Kinase Buffer B, perform the ligation
reaction using the 2×Ligation Mix after phenol/chloroform/isoamyl alcohol extraction andethanol precipitation.
-5-

2. Study of the molar ratio of inserts in ligation.
The vector : insert ratio in ligation greatly affects the ligation efficiency.

The table below

shows the molar ratio at which the best result was obtained when insert DNA of variouslengths was ligated and used for transformation in blunt-end ligation.
・ Blunt end
Insert length
VectorInsert200 bp15600 bp
151000 bp
12-103000 bp
10.5-2Vector： pUC19 ( 0.03 pmol ) cleaved with Sma I
Insert： Insert DNA cleaved with Sma I(0.015 pmol, 0.03 pmol, 0.06 pmol, 0.15 pmol, 0.3 pmol)
Ligation reaction： 16°C, 5 min

A word of caution in ligation reaction
Extended ligation, e.g., 16 hours or overnight, may cause a marked reduction intransformation efficiency.
When DNA is dissolved in a high salt concentration buffer, the ligation efficiency ismarkedly reduced. Prepare the DNA solution in ddH2O or TE buffer (pH 8.0).
The volume of the reaction solution used for transformation should be not more than
1/10 of the volume of the competent cells. Using too much reaction solution may
cause a reduction in transformation efficiency.
If the volume of the reaction solution is not less than 1/10 of the volume of thecompetent cells, recover the DNA after the ligation reaction by ethanol precipitation,
dissolve the DNA in ddH2O so that the volume of the DNA is not more than 1/10 of thevolume of the competent cells, and then perform transformation.
Ligation efficiency may vary depending on differences in the purity of DNA used for theligation reaction and the restriction enzymes to be used.
The volume of the reaction solution used for packaging should be not more than 1/10 ofthe volume of the packaging extract.

Using too much reaction solution may cause areduction in packaging efficiency.
If the volume of the reaction solution is not less than 1/10 volume of the packaging
extract, recover DNA after the ligation reaction by ethanol precipitation, dissolve theDNA in ddH2O so that the volume of the DNA is not more than 1/10 volume of thepackaging extract, and then perform transformation.
Packaging is not inhibited by using Gigapack（Stratagene Inc.）-6-NIPPON GENE CO., LTD.
2-7-18, TOIYA-MACHI, TOYAMA
930-0834 JAPANTel+81-76-451-6548
URL https://www.nippongene.com/

Blunting-Convenience Kit Manual (ver.2) 120831KH-7-