Курсовая работа: Vectors for moleculars cloning
Most of the lambda strains are stable for several years when stored at 4°C in SM buffer containing 0.3% freshly distilled chloroform (94). The master stocks of bacteriophage lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at -70°C for long-term storage. Klinman and Cohen have developed a method for storage of a phage library at -70°C by using top agar containing 30% glycerol.
Conclusion
In my work I determined investigations in Molecular cloning, familiarized with vectors for molecular cloning, summarized the received information and made consequences of scientists researches, defined the main tasks of molecular cloning, and made such conclusions:
1. sequences that permit the propagation of itself in bacteria (or in yeast for YACs) .
2. a cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut by many restriction enzymes .
3. a method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA; uually accomplished by selectable markers for drug resistance .
Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a bacterial (or yeast) cell and produces many copies of itself and the foreign DNA .
General Steps of Cloning with Any Vector :
1. prepare the vector and DNA to be cloned by digestion with restriction enzymes to generate complementary ends ;
2. ligate the foreign DNA into the vector with the enzyme DNA ligase;
3. introduce the DNA into bacterial cells (or yeast cells for YACs) by transformation ;
4. select cells containing foreign DNA by screening for selectable markers (usually drug resistance);
Literature
1. Finbar Hayes The Function and Organization of Plasmids// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp. 1-18.
2. Mallory J. A. White and Wade A. Nichols Cosmid Packaging and Infection of E. coli// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp. 67-70
3. Tim S. Poulsen and Hans E. Johnsen BAC End Sequencing // Bacterial Artificial Chromosomes Volume 1: Library Construction, Physical Mapping, and Sequencing.- 2007. – vol.255 - pp.157-162.
4. Andrew Preston Choosing a Cloning Vector// E. coli Plasmid Vectors Methods and Applications.- 2007.- vol.235 – pp. 19-22.
5. Sambrook, J., Fritsch, E. F., and Maniatis, T. (eds.) (1989) Bacteriophage λvectors, in Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 2.3–2.125.
6. Srividya Swaminathan and Shyam K. Sharan Bacterial Artificial Chromosome Engineering// Bacterial Artificial Chromosomes Volume 2 :Functional Studies.- 2007. – vol.256 – pp. 089-106
7. www.Microbiologybytes.com
8. www.wikigenes.org
9. http:// plasmid.hms.harvard.edu
10. www. Bookrags.com/YAC