instructions

product code DNA Ligation System

RPN1507

Warning
For research use only.
Not recommended or intended
for diagnosis of disease in
humans or animals.
Do not use internally or
externally in humans or
animals.

i RPN1507PLPL Rev-A, 2004

Handling Page finder
Handling 2
Storage
Store at -15 ºC to -30 ºC
Components 3

Safety warnings and precautions 3

Description 4

Protocol 5

Additional information
Troubleshooting 10
Background and references 11
Product information 12
Related products 12

● 2
Components Safety warnings and precautions
Solution A: reaction Warning: For research use only.
buffer 3 x 1000 µl
Not recommended or intended for diagnosis of
Solution B: enzyme disease in humans or animals. Do not use
buffer 2 x 187.5 µl
internally or externally in humans or animals.
* The kit components are
for 50 reactions when All chemicals should be considered as
using 60 µl of Solution A potentially hazardous. We therefore
and 7.5 µl of Solution B recommend that this product is handled only
per reaction.
by those persons who have been trained in
laboratory techniques and that it is used in
Notes: accordance with the principles of good
1.Solution A may be laboratory practice. Wear suitable protective
thawed and mixed at
clothing such as laboratory overalls, safety
room temperature.
Solution B, which glasses and gloves. Care should be taken to
contains the T4 DNA avoid contact with skin or eyes. In the case
ligase, should be thawed of contact with skin or eyes wash
on ice gently mixed immediately with water. See material safety
before use. The solutions
data sheet(s) and/or safety statement(s) for
can be thawed and frozen
repeatedly. specific advice.
2.DNA ligation mixtures
can be applied directly to
agarose gel
electrophoresis. Ethanol
precipitation* is
recommended for the
concentration of the DNA
sample when using
polyacrylamide gel. Do
not directly extract the
ligation mixture with
phenol.

● 3
*Ethanol precipitation: Description
1. Add one-tenth volume
£M sodium acetate (pH The DNA Ligation System (50 reactions) is a
5.2) or one-twentieth simple two-component system for performing
volume of 5M NaCI, and DNA ligation rapidly. The kit uses T4 DNA
2-2.5 volume of ethanol ligase and an optimized buffer system.
into the reactant.
Conventional overnight incubations are no
2. Leave at -20 °C for 20
longer required due to the high efficiency of
min, or at -80 °C for 10
min. the ligation reaction. Various kinds of
ligation can be completed within 30
3. Collect DNA by
centrifugation at 4 °C. minutes. For general ligation reactions, this
When small amount of kit gives results in only three minutes. The
DNA is to be collected, reaction can proceed directly to bacterial
carrier may be useful for
transformation, as well as in vitro packaging
ethanol precipitation.
procedures without the need to purify the
DNA. The kit contains enough reagents to
perform 50 reactions.

● 4
 Protocol
A. Insertion of DNA
fragments into
❶ Procedure
plasmid vectors 1. Combine plasmid vector DNA (which has been
digested with an appropriate restriction
endonuclease and dephosphorylated by treatment
with alkaline phosphatase) and the DNA fragment
to be inserted in a total volume of 5-10 µl. We
recommend 100 mM Tris-HCI, pH7.6, 5 mM
MgCI2 for dissolving DNA, however TE buffer (10
mM Tris-HCI, pH8.0, 1 mM EDTA) could also be
used. Recommended amounts of DNA are vector:
insert = 0.03 pmol: 0.1 -0.3 pmol. (0.03 pmol of
pUC18 DNA corresponds to about 50 ng).

2, Add 4-8 volumes*1 of Solution A to the DNA

solution and mix thoroughly.

3. Add one volume of Solution B (5-10 µl) and mix

thoroughly.

4. Incubate at 16 °C for 30 minutes*2.

5. The ligation reaction mixture can be used

directly for transformation with E.coli competent
cells. When performing transformation immediately
after ligation, apply 10 µl of the ligation mixture to
100 µl of competent cells*3.

● 5
*1 Usually, 4 volumes of Solution A is sufficient
for DNAs that are suspended in Tris-HCI MgCI2
buffer. If other buffers are used or if the
concentration of DNA is high, use 8 volumes of
Solution A to obtain optimum efficiency.
*2 The reaction should be carried out at 16 °C.
Higher temperatures (>26 °C) will inhibit the
formation of circular DNA. If good results are not
obtained, the reaction can be extended overnight.
Results depend on the purity of DNA. If good
results are not obtained, an additional phenol
extraction/ethanol precipitation step of the DNA
often helps.
*3 A maximum of 20 µl of the ligation reactant
may be used to transform 100 µl of E.coli
competent cells. More of the ligation mixture may
decrease transformation efficiency. The mixture
should not be used directly in electroporation, in
which case, the DNA should be precipitated with
ethanol and dissolved in low salt buffers such as
TE.

● 6
Table 1 Transformation efficiencies (colonies per µg insert DNA)
Insert/vector ratios can go up to as high as 10.0. Better results are
obtained by using dephosphorylated vector.

Vector fragment insert/vector (molar ration)

0.1 0.3 1.0 3.0 10.0
DNA dephosphorylated 1.7 x 10 5.0 x 10 1.7 x 10 2.3 x 10 2.1 x 107
6 6 7 7

Ligation System
30 minutes phosphorylated 7.8 x 105 2.5 x 106 8.2 x 106 1.7 x 107 2.3 x 107

T4 DNA dephosphorylated 1.6 x 105 2.0 x 105 1.8 x 106 3.1 x 106 1.9 x 106
ligase
16 hours phosphorylated 4.6 x 105 1.0 x 106 1.9 x 106 5.0 x 106 1.2 x 107

B. Insertion of DNA 1. Combine 250 ng (0.01 pmol) of λ phage vector

into λ phage vectors
DNA (which has been digested with an appropriate
restriction endonuclease and, if preferred,
dephosphorylated by treatment with alkaline
phosphatase) and the DNA to be inserted (0.03-
0.1 pmol) in a total volume of 5-10 µl. Best
results are obtained in buffer containing 100 mM
Tris-HCI, pH 6, 5 mM MgCI2 and 300 mM NaCI.
The salt concentration is important for producing
yields of concatemeric λ DNA. Therefore, if TE
(10 mM Tris-HCI, pH 8.0, 1 mM EDTA) is used,
the solution should be supplemented to give a
final concentration of 300 mM NaCI.
2. Add one volume (5-10 µl) of Solution B to the
DNA solution and mix well.
3. Incubate at 26 °C*1 for 5-10 minutes*2
4. The ligation reaction mixture (up to 5 µl) can be
used directly in λ in vitro packaging reactions*3
*1 Ligations involving λ phage vector DNA work
better when carried out at 26 °C than at 16 °C.

● 7
 *2 Ligation reactions set up as above are usually
complete after 5-10 minutes of incubation. Longer
reactions will not increase ligation efficiency.
*3 Components of the ligation mixture will not
inhibit λ in vitro packaging reactions as long as
≤10% in the volume ratio to the packaging extract
of mixture are used with a standard packaging
extract. If more of the ligation mixture is to be
packaged in a single reaction, the DNA should be
ethanol precipitated and be dissolved in TE buffer
in the volume ration of ≤10% to the packaging
extract in order to reduce the volume.

● 8
Table 2 Transformation efficiencies (white plaques per µg λgt11 DNA)

Incubation time DNA Ligation System T4 DNA Ligase

10 min 8.5 x 106 1.8 x 106

16 hr - 3.1 x 106

C. Self-circularization The procedure for self-circularization of linear DNA

of linear DNA is essentially the same as for A. (Insertion of DNA
(Intramolecular fragments into plasmid vectors). However, it is
important to use low concentrations of DNA in the
ligation)
ligation reaction to maximize intramolecular ligation
as well as to keep the volume of the DNA solution
low for higher bacterial transformation efficiency.

D. Linker [Adaptor] 1. Insertion of linker into a plasmid vector

ligation Conditions for linker ligation (8 bases or longer) are
essentially the same as for A. (Insertion of DNA
fragments into plasmid vectors). However, if the
linker is shorter than 8 bases or if the linker has a
low GC-content, the ligation reaction should be
carried out at 4-10 °C for 1-2 hours. Recommended
vector/linker molar ratios are:

● phosphorylated linker: dephosphorylated

vector = 10 ~100:1

● phosphorylated linker: dephosphorylated

vector = >100:1

2. Linker [Adaptor] ligation to both termini of a

DNA fragment (ex. Linker ligation of cDNA)

1. Prepare 5-10 µl of DNA solution containing

DNA fragment to be ligated (0.01-0.1 pmol) and
linker (or adaptor). Recommended DNA
fragment/linker [adaptor] molar ratio is:

● 9

Troubleshooting guide
Problems
❶
Ligation efficiency is low.
❷
Can ligation mixture be
directly used in
electroporation
● 10
● DNA fragment: linker [adaptor] = 1: >100
2. Add one volume (5-10 µl) of Solution B and
mix well.
3. Incubate at 16 °C for 30 min. However, if the
linker is shorter than 8 bases or if the linker has
low Gc-content, the ligation reaction should be
carried out at 4-10 °C for 1 - 2 hours.
4. Inactivate T4 DNA ligase by heating at 70 °C
for 10 minutes.
5. In case of performing the digestion with
restriction endonucleases, precipitate the DNA
with ethanol prior to digestion.
Remedies
1. Try one of the following:
● Extend the reaction time to overnight.
● Prior to use in transformation, add NaCI to the
final concentration of 500 mM to the ligation
mixture. Addition of salt can increase transformation
efficiency.
When either of the above two operations does not
work to increase the efficiency, re-purification of
DNA is recommended.
2. Transformation efficiency may decrease when
directly applying the ligation reactant to
electroporation. In that case, the DNA should be
precipitated with ethanol and dissolved in
appropriate buffer before use in electroporation.
Background and references
1. Hayashi, K, Nakazawa, M., Ishizaki, Y., Hiraoka, N. and Obayashi,
A Nucleic Acid Res., 14, 7617-7631. (1986)

● 11
Legal Product information
Amersham Biosciences
UK Limited, a General Product name code
Electric company, going to
market as GE Healthcare DNA Ligation System RPN1507

Amersham and
Amersham Biosciences
are trademarks of
Amersham plc Related products

5’ End Labelling Kit RPN1509

DNA Blunting and Ligation

System RPN1508

General Electric Company

reserves the right to make
changes in specifications
and features shown
herein, or discontinue the
product described at any http://www.amershambiosciences.com

time without notice or Amersham Biosciences UK Limited

obligation. Contact your Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK
Amersham Biosciences AB
GE Representative for the
SE-751 84 Uppsala Sweden
most current information.
Amersham Biosciences Corp
800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA
© 2004 General Electric Amersham Biosciences Europe GmbH
Company – All rights Munzinger Strasse 9 D-79111 Freiburg Germany

reserved

● 12