I. Description

II. Protocols

A. Insertion of DNA into Plasmid Vectors
B. Insertion of DNA into l Phage Vectors
C. Self-Circularization of Linear DNA (Intramolecular Ligation)
D. Linker (Adaptor) Ligation

III. Frequently Asked Questions

IV. Applications

A. One Hour Protocol For Subcloning and Size-Fractionated Deletion Cloning
B. Three-Minute Ligation

I. Description

The DNA Ligation Kit is a simple two-component system that produces very rapid ligation reactions using T4 DNA Ligase and an optimized buffer system. Due to the high efficiency of the ligation reaction, conventional overnight incubations are no longer required. Some ligations can be completed within 30 minutes, and in many situations an incubation time of three minutes is sufficient. The reaction mixture can be used directly for bacterial transformation and in vitro packaging procedures without further purification.

II. Protocols

A. Insertion of DNA Into Plasmid Vectors

1. Digest plasmid DNA with an appropriate restriction enzyme and dephosphorylate by treatment with alkaline phosphatase. Combine plasmid DNA and DNA to be inserted in a total volume of 5-10 µl. We recommend 100 mM Tris-HCl (pH 7.6), 5 mM MgCl₂ for resuspending DNA; however, TE buffer [10 mM Tris-HCI (pH 8.0), 1 mM EDTA] could also be used. Use 0.03 pmol of vector DNA (50 ng pUC18) and 0.1-0.3 pmol of insert.

2. Add 4-8 volumes^a of Solution A to the DNA solution and mix thoroughly.

3. Add 1 volume of Solution B (5-10 ml) and mix thoroughly.

4. Incubate at 16°C for 30 minutes.^b

5. The ligation reaction mixture can be used directly for bacterial transformation. Use 10-20 µl of the ligation mixture with 100 ml of competent bacterial cells.^c

Notes:

^a Usually, 4 volumes of Solution A are sufficient for DNA suspended in Tris-HCl, MgCl₂ buffer. If other buffers are used or the concentration of DNA is high, use 8 volumes of Solution A to obtain optimum efficiency.
^b 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. An additional phenol extraction/ethanol precipitation step may be needed if DNA purity is a problem.
^c A maximum of 20 µl of the ligation mixture may be used to transform 100 µl of competent bacterial cells. Excess ligation mixture may decrease transformation efficiency. For use in electroporation, DNA should be precipitated with ethanol and suspended in a low-salt buffer such as TE.

Example

In this experiment, 50 ng of EcoR I-digested pUC118 (25 fmol) were mixed with 2.5-250 ng (2.5-250 fmol) of a 1.5 kb EcoR I DNA fragment in total volumes of 5 µl. Solution A (25 µl) and Solution B (5 µl) were added to each DNA mixture and reactions were incubated at 16°C for 30 minutes. A portion of each solution was used directly to transform competent JM109 cells and transformation mixtures were spread onto L-Amp plates containing X-Gal and IPTG. Competent JM109 cells had a transformation efficiency of at least 6.3 x 107 transformants per µg of DNA when assayed with supercoiled pUC118 DNA. The insert:vector molar ratio can be as high as 10.0. Better results were obtained using dephosphorylated vector.

B. Insertion of DNA Into l Phage Vectors

1. Digest l phage vector DNA with an appropriate restriction enzyme and dephosphorylate using alkaline phosphatase if desired. Combine 250 ng (0.01 pmol) of the treated l DNA with the DNA to be inserted (0.03-0.1 pmol) in a total volume of 5-10 µl. Best results are obtained when DNA is resuspended in buffer containing 100 mM Tris-HCl (pH 7.6), 5 mM MgCl₂ and 300 mM NaCl. The salt concentration is important for producing concatemeric l DNA. If TE [10 mM Tris-HCl (pH 8.0), 1 mM EDTA] is used, the solution should be supplemented to give a final concentration of 300 mM NaCl.

2. Add 1 volume (5-10 µl) of Solution B to the DNA solution and mix well.

3. Incubate at 26°C^a for 5-10 minutes^b.

4. The ligation reaction mixture (up to 5 µl) can be used directly in l in vitro packaging reactions.^c

Notes:

^a Ligations involving l phage vector DNA work better when carried out at 26°C than at 16°C.
^b Ligation reactions set up as above are usually complete after 5-10 minutes of incubation.
Longer reactions will not increase ligation efficiency.
^c Components of the ligation mixture will not inhibit l in vitro packaging reactions as long as the ligation mixture constitutes <10% of the total volume when using a standard packaging extract.
If more of the ligation mixture is to be packaged in a single reaction, DNA should be ethanol precipitated and resuspended in TE buffer to reduce the volume.

Example

EcoR I-digested pBR322 DNA (25 ng) and EcoR I-digested, dephosphorylated lgt11 arms (250 ng) were combined in a total volume of 7.5 µl. One volume of Solution B was added and the reaction was incubated at 26°C for 10 minutes. Ligated DNA was packaged using a commercially available l in vitro packaging kit and was used to transfect E. coli Y1090
cells (r-).

C. Self-Circularization of Linear DNA (Intramolecular Ligation)

The procedure for self-circularization of linear DNA is essentially the same as for insertion of DNA fragments into a plasmid vector. However, it is important to use low concentrations of DNA while maintaining a small volume. Low concentrations of DNA in the ligation reaction maximize intramolecular ligation. Small volumes allow for higher bacterial transformation efficiency.

Example

pBR322 DNA was digested with Sca I. Solution A (40 µl) and Solution B (5 µl) were added to digested pBR322 DNA (350ng/10µl) and the reaction was incubated at 16°C for 30 minutes. The reaction solution was used directly to transform competent E. coli HB101 cells. Competent HB101 cells had a transformation efficiency of at least 1 x 108 transformants per µg of DNA when assayed with supercoiled pBR322 DNA.

D. Linker (Adaptor) Ligation

1. Insertion of linker into a plasmid vector.

Conditions for linker ligation (8 bases or longer) are essentially the same as for insertion of DNA fragments into a plasmid vector (see page 3). However, if the linker is shorter than 8 bases or has a low G+C content, the ligation reaction should be carried out at 4-10°C for 1-2 hours. The recommended molar ratio of phosphorylated linker:dephosphorylated vector is 10-100:1. When using phosphorylated vector, the linker:vector molar ratio should be >100:1.

2. Linker ligation to both termini of a DNA fragment (e.g., ligation of linkers to cDNA).
a. Combine the DNA fragment (0.01-0.1 pmol) and linker in a volume of 5-10 µl. The recommended linker:DNA fragment molar ratio is >100:1.
b. Add 1 volume (5-10 µl) of Solution B and mix well.
b. Incubate at 16°C for 30 minutes. If the linker is shorter than 8 bases or has a low G+C content the ligation reaction should be carried out at 4-10°C for 1-2 hours.

Following linker ligation, DNA may be digested directly with restriction endonucleases after inactivating the T4 DNA ligase (heating at 70°C for 10 minutes). If the restriction enzyme being used is known to display star activity, DNA can be precipitated with ethanol prior to digestion.

III. Frequently Asked Questions

Q1: What can be done if the ligation efficiency is low?

A1: • Extend the reaction time to overnight.
• Add salt to increase the transformation efficiency. Adjust the ligation mixture to a
final concentration of 500 mM NaCl prior to use in transformation.
• If the ligation efficiency is still low, re-purification of DNA is recommended.

Q2: Can the ligation mixture be used directly for electroporation?

A2: Transformation efficiency may decrease when using the ligation mixture for electroporation.
In this case, the DNA should be ethanol precipitated and resuspended in a low salt buffer prior to electroporation.

Q3: Can the reaction products be analyzed by gel electrophoresis?

A3: No further purification of the ligation mixture is needed when using an agarose gel. Ethanol precipitation* is recommended for concentrating the DNA sample when using a polyacrylamide gel. Do not extract the ligation mixture with phenol.

*Ethanol precipitation:

1) Add 1/20 volume of 3 M NaCl and 2-2.5 volumes of ethanol to the ligated DNA.

2) Incubate at -20°C for 20 minutes or at -80°C for 10 minutes.

3) Collect DNA by centrifugation at 4°C. When small amounts of DNA are to be collected, carrier RNA or glycogen may be added to the ethanol precipitation mixture.

IV. Applications
A. One Hour Protocol For Subcloning and Size-Fractionated Deletion Cloning

The recovery of DNA fragments from agarose gel slices and the cloning of these fragments into vector DNA can be accomplished rapidly by the combined use of SUPREC™-01 DNA Extraction Cartridge, SUPREC-02 DNA Purification System, and a DNA Ligation Kit. Only a small amount of DNA is required because of the high efficiency of the procedure.

Step 1 Electrophoretic Separation of DNA

· DNA Restriction Fragments: Digest 1-2 µg of DNA with restriction enzyme and separate by agarose gel electrophoresis. Excise the slice of agarose that contains the DNA band of interest.

· Serial Deletion Fragments: Use 5 µg of DNA with the Deletion Kit (TAK 6030) and separate by electrophoresis. Excise the gel to obtain fragments truncated to the desired size range.

Step 2 SUPREC-01 DNA Extraction Cartridge (5 minutes)

Insert the agarose gel slice (50-100 µl) into the SUPREC-01 DNA Extraction Cartridge and centrifuge at 10,000 rpm in a microcentrifuge for 5 minutes.

Step 3 SUPREC-02 DNA Purification System (3 minutes)

Transfer the filtrate to the SUPREC-02 DNA Purification System cartridge and centrifuge at 6,000 rpm in a microcentrifuge for 3 minutes. Add 15 µl of TE buffer, pipet well, and collect DNA from the cartridge filter.

Step 4 Ligation (15 minutes)

Use 10 µl of DNA solution for ligation at room temperature (16-25°C) for 15 minutes using the DNA Ligation Kit.

Step 5 Transformation (31 minutes)

Mix 10 µl of the ligation reaction solution with 100 µl of competent cells and incubate on ice for 15 minutes followed by heat shock at 42°C for 30 seconds. Add 890 µl of SOC media and incubate at 37°C for 15 min.

Step 6 Plating (Total - 60 minutes)

Plate on selective media.

B. Three-Minute Ligation

The following examples demonstrate that a three-minute ligation at 25°C produces results similar to ligations performed under conventional conditions (30 minutes at 16°C).

Self-Ligation of Linearized DNA (Sticky- and Blunt-End Ligation)

pUC118 DNA (200 ng/10 µl) was digested with either EcoR I or Hinc II, then recircularized using the DNA Ligation Kit. Ligations were performed at 25°C for 3 minutes and at 16°C for 30 minutes. A portion of each ligation reaction (16 ng/1.6 µl) was used to transform competent
E. coli JM109 cells (1.3 x 108 transformants/µg supercoiled pUC118 DNA).

Linker Ligation

pUC118 DNA was digested with Hinc II and dephosphorylated with alkaline phosphatase. Treated pUC118 (100 ng) was ligated to 260 ng of phosphorylated Bgl II linkers
(5'-CAGATCTG-3') using the DNA Ligation Kit. Ligations were incubated at either 25°C for 3 minutes or 16°C for 30 minutes. Part of each ligation reaction was used directly to transform competent E. coli JM109 cells (1.3 x 108 transformants/µg supercoiled pUC118 DNA).