Cloning Reagents
Cloning is a technique in which recombinant DNA is inserted into a host cell and the amount of target gene is increased to obtain many DNA fragments. The gene cloning has greatly contributed to the development of molecular biology and gene recombination technology. A variety of processes are required for gene cloning, including insertion of DNA fragments into a cloning vector and amplification using host cells. Fujifilm Wako offers reagents necessary for a series of processes of cloning.
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Selection of a Gene Cloning Method
For cloning, sticky-end cloning, which uses a vector and inserts DNA digested with two (or one) restriction enzymes, is often chosen due to its easy operation and high efficiency. Amplification products obtained using a PCR enzyme with terminal transferase activity, such as Taq DNA polymerase, have single A added to the 3' end of double-stranded DNA. For these products, TA cloning using a T-vector is an effective option.
On the other hand, when amplification products obtained using a PCR enzyme with proofreading activity that produces blunt ends are used, sticky-end cloning using ligation of linkers with a restriction enzyme site or blunt-end cloning using blunt-end vector DNA is selected. In blunt-end cloning, ligation can be performed without dependence on sequence if the DNA ends are blunt, in the absence of suitable restriction sites in target gene and vector DNA.
History of Transformation using Competent Cells
In 1970, Mandel and Higa found that treatment with calcium chloride allowed Escherichia coli (E. coli) to take up DNA from bacteriophage λ1）. This was the first example of transformation of E. coli. Later, in 1973, Choen, Chang, Hsu, et al. demonstrated that this method was also effective for plasmid DNA2）.
Since then, transformation of E. coli has become a fundamental and important means of genetic engineering research, and various improvements have been tried. Methods for preparing competent E. coli using treatments other than calcium chloride, including monovalent cation3）, DMSO3）, DTT3）, or PEG4） have been reported. Following these protocols, 107 ～ 109 transformants can be obtained per microgram of plasmid.
Preparation of competent E. coli for experiment every time is time-consuming and technique-required. Also, even if highly competent E. coli are obtained, they are not easily stored in stable condition for a long time. The use of commercially available, freeze-thaw resistant, long-term storable competent cells enables achievement of high and stable transformation efficiencies.
References
- Mandel, M. and Higa, A. : J. Mol. Biol ., 53(1), 159 (1970).
Calcium-dependent bacteriophage DNA infection - Cohen, S. N., Chang, A. C. Y. and Hsu, L. : Proc. Natl. Acas. Sci. USA, 69(8), 2110 (1973).
Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA - Hanahan, D. : J. Mol. Biol ., 166(4), 557 (1983).
Studies on transformation of Escherichia coli with plasmids - Chung, C. T., Niemela, S. L. and Miller, R. H. : Proc. Natl. Acad. Sci. USA, 86(7), 2172 (1989).
One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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