Cat. #
1400
TaKaRa Restriction Enzyme
Starter BOX
Product Manual
v201108Da
Cat. #1400
TaKaRa Restriction Enzyme Starter BOX
v1108Da
Contents of TaKaRa Restriction Enzyme Starter BOX
Restriction Enzyme(13 total)
Bam H I
Eco R I
Hin d III
Kpn I
Nde I
Not I
Pst I
1,000 U
1,000 U
1,000 U
500 U
200 U
200 U
1,000 U
Sac I
Sal I
Sma I
Sph I
Xba I
Xho I
500 U
1,000 U
500 U
200 U
1,000 U
500 U
Ligation Kit
DNA Ligation Kit <Mighty Mix>
150 μl
DNA Marker
100 bp DNA Ladder (Dye Plus)
200 μl
1 Set
Universal Buffer Set
Universal Buffer Set
10X L, 10X M, 10X K, 10X T (BSA-free),
10X H
1 ml/tube
1 tube each
2 tubes
1 ml
10X Loading Buffer
Storage － 20℃
Compositions of Universal Buffer (Stored at -20℃)
1．10X L
2．10X M
3．10X H
100 mM
100 mM
10 mM
100 mM
100 mM
10 mM
500 mM
500 mM
100 mM
10 mM
1,000 mM
Tris-HCl, pH7.5
MgCl2
Dithiothreitol
Tris-HCl, pH7.5
MgCl2
Dithiothreitol
NaCl
Tris-HCl, pH7.5
MgCl2
Dithiothreitol
NaCl
4．10X K
5．10X T
(BSA-free)
200 mM
100 mM
10 mM
1,000 mM
330 mM
100 mM
5 mM
660 mM
6．0.1％ BSA
7．0.1％ Triton X-100
Tris-HCl, pH8.5
MgCl2
Dithiothreitol
KCl
Tris-Ac, pH7.9
Mg-Ac
Dithiothreitol
K-Ac
Compositions of 10X Loading Buffer (Store at RT after use)
0.09％
50％
0.05％
SDS
Glycerol
Bromophenol Blue
Add >1/10 volume of 10X Loading Buffer to stop enzyme reaction and load on agarose gel
for electrophoresis. SDS may precipitate during storage at room temperature. If precipates
are present, then dissolve in warm bath before use.
2
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Cat. #1400
TaKaRa Restriction Enzyme Starter BOX
Bam H I
Reaction Buffer
v1108Da
G G A T C C
C C T A G G
Volume
Conc.
1,000 units
15 units/μl
10X K Buffer
General Reaction Mixture
Bam H I
10X K Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 30℃
＊This enzyme shows equivalent activity at 37℃ compared to 30℃, but enzyme stability in
reaction mixture at 37℃ is lower than that of 30℃.
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Universal Buffer
Relative Activity (%)
L
(<20)
M
<20
H
40
K
100
T(+BSA)
(<20)
Effect of DNA Methylation
Enzyme activity is not affected by dam methylase, dcm methylase and CG methylase.
Star Activity
Unrelated site may often be cut in the presence of a a high concentration of glycerol or Mn 2+,
and at low ionic strength.
Eco R I
Reaction Buffer
G A A T T C
C T T A A G
Volume
Conc.
1,000 units
15 units/μl
10X H Buffer
General Reaction Mixture
Eco R I
10X H Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Relative Activity (%)
L
(20)
M
(100)
H
100
K
(120)
T(+BSA)
(80)
Effect of DNA Methylation
Enzyme activity is affected by CG methylase depending on the sequence following the recognition site.
Star Activity
Unrelated sites may often be cut in the presence of a a high concentration of glycerol or Mn2+,
and at low ionic strength. The addition of spermine (ca. 0.2 mM) reduces star activity to 30 50%, but also reduces enzyme activity to 70 - 80%.
URL:http://www.takara-bio.com
3
Cat. 1400
TaKaRa Restriction Enzyme Starter BOX
Hin d III
Reaction Buffer
v1108Da
A A G C T T
T T C G A A
Volume
Conc.
1,000 units
15 units/μl
10X M Buffer
General Reaction Mixture
Hin d III
10X M Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (70℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Relative Activity (%)
L
(60)
M
100
H
<20
K
200
T(+BSA)
(100)
Star Activity
Unrelated sites may often be cut in the presence of Mn2+.
Kpn I
Reaction Buffer
G G T A C C
C C A T G G
Volume
Conc.
500 units
10 units/μl
10X L Buffer
General Reaction Mixture
Kpn I
10X L Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Relative Activity (%)
L
100
M
60
H
<20
K
<20
T(+BSA)
(100)
Effect of DNA Methylation
Enzyme activity is not affected by CG methylase.
Notice
4
A long reaction time using excess enzyme should be avoided, since this enzyme tends to show
star activity.
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Cat. #1400
TaKaRa Restriction Enzyme Starter BOX
Nde I
Reaction Buffer
v1108Da
C A T A T G
G T A T A C
Volume
Conc.
200 units
8 units/μl
10X H Buffer
General Reaction Mixture
Nde I
10X H Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (70℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers
Relative Activity (%)
L
<20
M
40
H
100
K
100
T(+BSA)
80
Ligation Recutting Test
Ligation efficiency of DNA fragments with sticky ends generated by this enzyme is lower than
general 2-base blunt ends. Therefore, more efficient ligation can be achieved by using the
reaction conditions for blunt end ligation.
Notice
Do not dilute, as this enzyme is unstable at low concentration. Addition of Triton-X 100 to a final
concentration of 0.01% in reaction mixture increases the relative activity up to approx. 150%.
Not I
Reaction Buffer
G C G G C C G C
C G C C G G C G
Volume
Conc.
200 units
10 units/μl
10X H Buffer, 0.1％ BSA, 0.1％ Triton X-100
General Reaction Mixture
Not I
10X H Buffer
0.1％ BSA
0.1％ Triton X-100
Substrate DNA
Sterilized distilled water
1 μl
2 μl
2 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Relative Activity (%)
L
(<20)
M
(<20)
H
20 ＊
K
<20
T(+BSA)
(<20)
＊：100% activity is obtained by addition of both 0.01% BSA and 0.01% Triton X-100.
Effect of DNA Methylation
Enzyme activity is affected by CG methylase.
URL:http://www.takara-bio.com
5
Cat. 1400
TaKaRa Restriction Enzyme Starter BOX
Pst I
Reaction Buffer
v1108Da
C T G C A G
G A C G T C
1,000 units
Volume
15 units/μl
Conc.
10X H Buffer
General Reaction Mixture
Pst I
10X H Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
L
(<20)
Relative Activity (%)
M
(60)
H
100
K
80
T(+BSA)
(20)
Star Activity
Unrelated sites may often be cut in the presence of a a high concentration of glycerol.
Sac I
Reaction Buffer
G A G C T C
C T C G A G
Volume
Conc.
500 units
10 units/μl
10X L Buffer
General Reaction Mixture
Sac I
10X L Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers
Relative Activity (%)
L
100
M
60
H
<20
K
<20
T(+BSA)
80
Effect of DNA Methylation
Enzyme activity is not affected by CG methylase.
Star Activity
Unrelated sites may often be cut in the presence of a a high concentration of glycerol.
6
URL:http://www.takara-bio.com
Cat. #1400
TaKaRa Restriction Enzyme Starter BOX
Sal I
Reaction Buffer
v1108Da
G T C G A C
C A G C T G
Volume
Conc.
1,000 units
15 units/μl
10X H Buffer
General Reaction Mixture
Sal I
10X H Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Relative Activity (%)
L
<20
M
<20
H
100
K
(20)
T(+BSA)
<20
Effect of DNA Methylation
Enzyme activity is affected by CG methylase.
Star Activity
Unrelated sites may often be cut in the presence of a a high concentration of glycerol.
Sma I
Reaction Buffer
C C C G G G
G G G C C C
Volume
Conc.
500 units
10 units/μl
10X T Buffer, 0.1％ BSA
General Reaction Mixture
Sma I
10X T Buffer
0.1％ BSA
Substrate DNA
Sterilized distilled water
1 μl
2 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 30℃
＊This enzyme shows equivalent activity at 37℃ compared to 30℃, but enzyme stability in
reaction mixture at 37℃ is lower than that of 30℃ .
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers
Relative Activity (%)
L
<20
M
<20
H
<20
K
<20
T(+BSA)
100
Effect of DNA Methylation
Enzyme activity is affected by CG methylase.
URL:http://www.takara-bio.com
7
Cat. 1400
TaKaRa Restriction Enzyme Starter BOX
Sph I
Reaction Buffer
v1108Da
G C A T G C
C G T A C G
Volume
Conc.
200 units
10 units/μl
10X H Buffer
General Reaction Mixture
Sph I
10X H Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers　( ) : Weak star activity is detected.
Relative Activity (%)
L
(20)
M
(40)
H
100
K
120
T(+BSA)
(20)
Effect of DNA Methylation
Enzyme activity is not affected by CG methylase.
Xba I
Reaction Buffer
T C T A G A
A G A T C T
Volume
Conc.
1,000 units
15 units/μl
10X M Buffer, 0.1％ BSA
General Reaction Mixture
Xba I
10X M Buffer
0.1％ BSA
Substrate DNA
Sterilized distilled water
1 μl
2 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition
Heat treatment (60℃ , 15 minutes) or Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers
Relative Activity (%)
L
<20
M
80 ＊
H
20
K
<20
T(+BSA)
120
＊：100% activity is obtained by addition of 0.01% BSA.
Effect of DNA Methylation
When the sequence includes the recognition site, TCTAGATC, the enzyme activity is affected
by dam methylase. In this case, general DNA originated fom E. coli can not be cleaved by this
enzyme.
Star Activity
Unrelated sites may often be cut in the presence of a high concentration of glycerol.
8
URL:http://www.takara-bio.com
Cat. #1400
TaKaRa Restriction Enzyme Starter BOX
Xho I
Reaction Buffer
v1108Da
C T C G A G
G A G C T C
Volume
Conc.
500 units
10 units/μl
10X H Buffer
General Reaction Mixture
Xho I
10X H Buffer
Substrate DNA
Sterilized distilled water
1 μl
2 μl
≦ 1 μg
up to 20 μl
Reaction Temperature 37℃
Inactivation Condition Phenol treatment/ethanol precipitation
Relative Activity in TaKaRa’s Universal Buffers
Relative Activity (%)
L
<20
M
60
H
100
K
160
T(+BSA)
60
Effect of DNA Methylation
Enzyme activity is affected by CG methylase. (It cleaves CT5mCGAG very slowly.)
Ligation-Recutting Test
Ligation efficiency of DNA fragments with sticky ends generated by this enzyme is lower than
general 4-base blunt ends. Therefore, more efficient ligation can be achieved by using the
reaction conditions for blunt end ligation.
URL:http://www.takara-bio.com
9
TaKaRa Restriction Enzyme Starter BOX
Cat. #1400
v1108Da
DNA Ligation Kit <Mighty Mix>
Components
Ligation Mix
150 μl
20 rxn (when 7.5 μl used per reaction)
Storage
－ 20℃
Before using the kit, thaw on ice (for 5 - 10 min.) and mix thoroughly by pipetting.
The performance of the DNA Ligation Mix was confirmed to be unaffected up to at
least 50 freeze-thaw cycles.
Procedure
Insertion of DNA fragments into plasmid vectors
The Standard Protocol should be used for general ligation reactions. When performing
sticky-ended DNA ligations or when the highest efficiencies are not required, the Rapid
Protocol offers good efficiency in a shorter period of time.
[Standard Protocol]
1. Combine the digested plasmid vector DNA and the DNA fragment to be inserted in a
total volume of 5 - 10 μl. We recommend using TE buffer (10 mM Tris- HCI, pH 8.0, 1 mM
EDTA) for dissolving DNA. The recommended vector : insert ratio is 25 fmol vector : 25
- 250 fmol insert. (Note : 25 fmol of pUC118 DNA (3,162 bp) corresponds to about approx. 50 ng).
2. Add one volume of Ligation Mix (5 - 10 μl) to the DNA solution and mix thoroughly.
3. Incubate at 16℃ for 30 minutes＊1.
4. The ligation reaction mixture can be used directly for transformation with E.coli competent cells. When performing transformation immediately following ligation, add 10 μl
of the ligation mixture to 100 μl of competent cells＊2,3.
＊1 : If good results are not obtained, the reaction can be extended overnight.
＊2 : If more than 10 μl of the ligation reaction mixture must be used for transformation, then the ligated DNA should instead be ethanol precipitated prior to use.
＊3 : The ligation reaction mixture should not be used directly in electroporation.
Instead, ligated DNA should be ethanol precipitated and dissolved in a low
Salt buffer such as TE buffer prior to use.
[Rapid Protocol]
1. Combine the digested plasmid vector and the DNA fragment to be inserted in a total
volume of 5 - 10 μl. We recommend using TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM
EDTA) for dissolving DNA. Recommended vector : insert ratios are 25 fmol vector : 25
- 250 fmol insert (Note : 25 fmol of pUC118 DNA (3,162 bp) corresponds to about approx. 50 ng).
2. Add one volume of Ligation Mix (5 - 10 μl) to the DNA solution and mix thoroughly.
3. Incubate at 25℃ for 5 minutes＊1.
4. The ligation reaction mixture can be used directly for transformation with E.coli competent cells. When performing transformation immediately after ligation, add 10 μl of
the ligation mixture to 100 μl of competent cells＊2,3.
＊1 : Higher temperatures (>26℃) will inhibit the formation of circular DNA. The reaction temperature should be strictly kept at 25℃ when using the Rapid Protocol.
＊2 : If more than 10 μl of the ligation reaction mixture must be used for transformation, the ligated DNA should instead be ethanol precipitated prior to use.
＊3 : The ligation reaction mixture should not be used directly in electroporation.
Instead, ligated DNA should be ethanol precipitated and dissolved in a low
Salt buffer such as TE buffer prior to use.
10
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TaKaRa Restriction Enzyme Starter BOX
Cat. #1400
v1108Da
100 bp DNA Ladder (Dye Plus)
Volume
200 μl(for 40 lanes)
Storage
－ 20℃
Once opened or thawed, store at 4℃ and use sooner to avoid contamination.
Description This marker consists of 10 fragments between 100 bp and 1000 bp in multiples of 100 bp
and an additional fragment at 1500 bp. The 500 bp band containing triple the mass of
the other fragments, serves as a visible reference indicator; all other fragments appear
with equal intensity on the gel. They are all double-stranded DNA fragments. Since
this product is premixed with loading dyes and glycerol, it is possible to apply this
product directly to a gel for electrophoresis.
Fragment
Size (bp)
A
1,500
B
1,000
C
900
D
800
E
700
F
600
G
500
H
400
I
300
J
200
K
100
Application example
Apply 5 μl of DNA Ladder on an agarose gel, such as 3% NuSieve® 3:1 Agarose (Lonza).
Perform staining with EtBr, SYBR® Green I Nucleic Acid Gel Stain or GelStar® Nucleic
Acid Stain (Lonza).
URL:http://www.takara-bio.com
11
TaKaRa Restriction Enzyme Starter BOX
Cat. #1400
v1108Da
NOTE : This product is for research use only. It is not intended for use in therapeutic or diagnostic
procedures for humans or animals. Also, do not use this product as food, cosmetic, or
household item, etc.
Takara products may not be resold or transferred, modified for resale or transfer, or used
to manufacture commercial products without written approval from TAKARA BIO INC.
If you require licenses for other use, please contact us by phone at +81 77 543 7247 or
from our website at www.takara-bio.com.
Your use of this product is also subject to compliance with any applicable licensing
requirements described on the product web page. It is your responsibility to review,
understand and adhere to any restrictions imposed by such statements.
12
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