BAC/PAC library construction
BAC/PAC library construction
BAC (Bacterial Artificial Chromosome) libraries can be used for (1) whole genome sequencing; (2) construction of physical maps, chromosome step-by-step; (3) gene screening; and (4) gene locus cloning.
Operation method
Construction of BAC libraries
Principle
BAC is a cloning vector system for loading large fragments of DNA, which is used for the construction of human, animal and plant genome libraries.BAC has the advantages of large insertion fragments (several thousand bases to 350kb), low chimerism, good genetic stability, easy to manipulate and so on.The construction of BAC libraries is an important foundation for the study of eukaryotic genomes with large genomes, and it can be used for physical mapping, gene structure and function analysis of the important genes and the whole genome of eukaryotes. It can be used for the physical mapping of important eukaryotic genes and the whole genome, the localization and cloning of important functional genes, and the analysis of gene structure and function.
Materials and Instruments
EcoR I-digested genomic DNA Vector DNA Electrotransformed Receptor Bacterial Cells Move 1. Size of target region (1) If the target region of the genome is very small (less than 50kb), then mucoid vectors or even λ phage vectors can be chosen. Using the existing commercialized mucoid vectors, efficient packaging mixtures and suitable E. coli strains, researchers can easily construct mucoid vector libraries. (2) If the target region of the genome is relatively large, then P1, PAC or BAC is more suitable; P1 is more difficult to operate, while PAC is relatively easy to operate; BAC vectors are also good choices, and the researcher can utilize existing mature products to construct BAC libraries, such as a series of BAC vectors and the supporting kits from EPICENTRE. (3) If the target region is very large (greater than 250kb), then YAC vector is the first choice. However, the application of YAC vectors to construct genomic libraries is very cumbersome and difficult to operate, and is usually done by specialized companies. 2. Difficulty of screening libraries Mucoid vectors are generally screened by traditional filter membrane photocopying hybridization, but this method is laborious and wasteful for the construction of region mapping and stacked clusters. Large-capacity vector libraries, such as P1, PAC, BAC, and YAC, are stored in two-dimensional arrays, which can be screened both by traditional single-copy probe hybridization and by PCR for population screening of multiple clones. Moreover, the use of high-capacity vectors to construct libraries can reduce the steps of chromosome step checking. 3. Consideration of vector copy number When large DNA fragments are cloned into high-copy vectors, the cloned DNA will be recombined, thus affecting the fidelity and stability of the cloned sequences; while the low yield DNA of single-copy vectors is a bottleneck for high-throughput analysis. This contradiction often troubles researchers. EPICENTRE's CopyControlTM cloning system is the best solution to these difficulties. copyControlTM technology combines the advantages of single-copy vectors and multi-copy vectors. The single-copy vector improves the stability of the inserted fragments, and the copy vector can instantly amplify high-copy-number clones under the action of an inducer to obtain a high yield of DNA. Ligation of genomic DNA fragments into the vector Use ligase to ligate the above DNA fragments of appropriate size into the vector. The commercial vector has been pre-treated and can be used directly without endonuclease digestion and dephosphorylation. The ligation system should be 100ul. Note: After ligating the BAC vector, the ligated product should be desalted to remove the salt in the ligation buffer. V. Transfection of recombinant vector into host cells 1. If the Epicentre kit is used to construct a plating library, use Epicentre MaxPlax Lambda Packaging Extracts to package the ligation products as described above, measure the titer of the packaged plating clone, and then transfect it with the Epicentre EPI300-T1 Plating Strain. The recombinants were singled out and the size of the insert fragments was identified. If the size and quality of the library is satisfactory, the plate can be spread for library screening, or amplify and preserve the library. 2. If the Epicentre kit is used to construct the BAC library, the electrocompetent method should be chosen. Epicentre TansforMaxTM EPI300TM Electrocompetent E.coli can be chosen as the host, and the above ligated product will be transferred into the bacteria, and the plate will be coated to grow the clone. Clones are obtained and the researcher needs to evaluate the size of the BAC clone to determine if the library size can meet the requirements.EPILyse and EPIBlue in the Epicentre Library Construction Kit, which quickly lyses the clones and electrophoreses them, can easily identify the size of the BAC clone, thus estimating the size of the BAC library. If the library size is appropriate, then this library is ready for subsequent operations. Determination of the number of clones in the library Genomic DNA libraries need to contain enough clones to ensure the representativeness of the library. Generally use the following empirical formula to determine: N = ln (1-P ) / ln (1-f ) P is the desired coverage, f is the ratio of insert fragment size to genomic DNA size, and N is the number of clones required. For example, if a human genome library is constructed with a BAC vector, the human genome size is 3 x 109 bp, the insert fragment size is 100 kb, and the desired coverage is 99%, then the number of BAC clones required is: N = ln (1 - 0.99) / ln (1 - [106bp / 3 x 109 bp]) = -4.61 / -3.33 x10-6= 138,298 For more product details, please visit Aladdin Scientific website.
5 X buffer EDTA Protein Hydrolase K PMSF buffer Not I restriction enzyme and buffer Agarose solution 0.5 X TBE buffer
SOC Medium LB Plate LB Medium Water Bath Radial Dialysis Membrane Centrifuge Tubes Orbital Mixer Automated Plasmid Isolation System Soft Plastic 96-well Plate