30 rxn

Description

Two crucial procedures in cloning are the ligation of the foreign DNAs to the vector DNA and the transformation of bacteria using those ligated DNA constructs (the recombinant molecules). Ligation is accomplished using the enzyme DNA ligase (usually from the bacteriophage T4). It requires ATP and magnesium ions to catalyze the reaction of a 3'-OH and a 5'-P on double-stranded DNA to form a phosphodiester bond. The DNA ends can be cohesive ends, such as those formed between molecules that have been digested with the same restriction endonuclease, or they can be blunt ends. Ligation between cohesive-ended molecules is much more efficient than ligation between blunt-ended molecules. Because of this, when ligatiog blunt-ended molecules, the DNA concentration must be higher than when ligating cohesive-ended molecules. The rates of blunt-end and cohesive-end ligation of DNA by T4 DNA ligase are increased by orders of magnitude in the presence of Polyethylene glycol (PEG) [Pheiffer, B. H., Zimmerman, S. B., Nucleic Acids Res., 11, 7853-7871, 1983]. The Rapid T4 DNA Ligase is based on this PEG-assisted rapid ligation of DNA and enables sticky- and blunt-ended DNA ligation in only 10 min. 5×Rapid Ligation Buffer (RLB) supplied in this kit is specially designed for the efficient rapid ligation of DNA. For an unknown reason, while ligation efficiency appears to increase for high PEG concentration, the transformation efficiency for molecules ligated in this condition decrease. Therefore, appropriate PEG concentration should be selected. At the PEG concentration should be selected. At the PEG concentration utilized in the Rapid T4 DNA Ligase, ligation and transformation are  both increased significantly.

Storage

All components should be stored at -20℃. Do not substitute the reaction buffer supplied in this kit with other one.

Contents

For research use only. The Rapid DNA Ligaton Kit contains sufficient reagents to perform approximately 30×20 μl DNA ligaton reactions.

                               Materials Provided                               Size (30 reactions )

                                  T4 DNA ligase                                        30 μl

                    5×Rapid Ligation Buffer (5×RLB)                     200 μl

Characteristics

Sufficient ligation efficiency can be archived at room temperature (20-25℃) for 10-30 min.

Storage Buffer

          10mM            Tris-HCl (pH 7.5)

          50mM            KCl

          1mM              Dithiothreitol

          50%              Glycerol

Quality Control

T4 DNA ligase supplied in this kit is free of detectable exo- or endonuclease activities. Each lot is functionally tested in the ligation reaction. Additionally, each lot is analyzed by SDS polyacrylamide gel electrophoresis for the presence of detectable contaminating proteins (less than 5%).

     Exonuclease assay:

After incubation of a 50 μl reaction containing T4 DNA ligase with 1 μg of sonicated [3H]-labeled E.coli DNA (105 cpm/μg) for 4 hours at 37℃. The DNA is precipitated with trichloroacetic acid and the radioactivity of the supernatant is evaluated. Exonuclease activity is expressed as a percent of total DNA radioactivity released into the acid soluble fraction. The exonuclease activity was determined to < 0.1% radioactivity. The limit of detectability of this assay is approximately 0.05%.

     Endonuclease assay:

Incubation of a 50 μl reaction containing T4 DNA ligase with 1 μg of supercoiled plasmid DNA for 4 hours at 37℃ resulted in < 0.1% conversion to nicked or linear plasmid DNA as determined by agarose gel electrophoresis.

Protocol

     A. Cloning of DNA fragments into plasmid vectors

We recommend using a 1:1 or 1:3 molar ratio of vector: insert DNA. These ratios will vary with the types of vectors, for example, cDNA and genomic DNA cloning vectors. To calculate the required amount of insert DNA, the following formula can be used.

[Example]

How much 0.5 kbp insert DNA should be added to a ligation in which 100 ng of 6 kbp vector will be used? The desired vector: insert ratio will be 1:3.  

1. Prepare the following reaction in a sterile microcentrifuge tube:

2. Incubate the reaction at room temperature for 10 min.

3. Competent E. coli was transformed with the ligation mixture.

     (Competent cell efficiency: > 1×10⁷)

     B. Recircularization of linear DNA

The amount of DNA is important in recircularization. For the enhanced recircularization, lower DNA concentration is helpful compared to the cloning of DNA fragment into plasmid vectors because low DNA concentration makes more intramolecular ligation (recircularization) and suppress intermolecular ligation. Intramolecular ligation is Concentration-independent.

[Example]

1. Prepare the following reaction in a sterile microcentrifuge tube:  

2. Incubate the reaction at room temperature for 10 min.

3. Competent E. coli was transformed with the ligation mixture.

     (Competent cell efficiency: > 1×10⁷)

     C. Adaptor ligation

Adaptor ligation conditions are basically the same as for cloning of DNA fragments into plasmid vectors (8 bases or longer). However, if the adaptor is shorter than 8 bases or if GC-contents are low in adaptors, the ligation reaction should be carried out at 16℃ for 30minutes to 2 hours. We recommended vector/linker molar ratio is: Dephosphorylated vector: Phosphorylated linker > 1:100

[Example]

1. Prepare the following reaction in a sterile microcentrifuge tube:

2. Incubate the reaction at 16℃ for 1 hour.

3. Competent E. coli was transformed with the ligation mixture.

       (Competent cell efficiency: >1×10⁷)

or

In the case of adaptor ligation to termini, inactivate T4 DNA ligase by heating at 70℃ for 10 min and appropriate purification is performed for user’s purpose.

There are many problems with ligation reaction itself and with the competent cells, the selection medium, the restriction endonuclease digestion of the vector, and the phosphatase treatment of the vector. Because transformation of some competent cells is inhibited by components of the ligation reaction, the reaction should be diluted fivefold before being used for transformation. Some possible causes of unsuccessful ligation are listed next along with suggested solutions.