BAC library construction methods and techniques
BAC library construction skills -------------------------------------------------- ------------------------------ I. Isolation of genomic DNA (gDNA) There is no doubt that quality genomic DNA is critical to the construction of genomic libraries. Researchers need to consult the literature and use experience to select the appropriate genomic DNA isolation method to ensure that the DNA is not excessively sheared or degraded during the separation process, while also ensuring the purity of the DNA. Second, processing genomic DNA According to the research purpose, researchers need to choose the appropriate carrier. Different vectors have different requirements for the length of genomic DNA, and researchers must select a suitable length of genomic DNA to construct a genomic library. requires attention: (1) When electrophoresis, the appropriate DNA Ladder should be selected to ensure accurate positioning of the DNA of the desired molecular weight after electrophoresis. When constructing a cosmid library using the Biochem Cryptology Kit, you can directly use the Control Insert DNA in the library kit to make the marker, which is convenient and accurate. (2) After electrophoresis, the target DNA should be avoided by ultraviolet light, and ultraviolet light irradiation can significantly reduce the cloning efficiency. Solution: Cut off the part of the gel where the marker is located, mark it under the UV lamp, and use this as a reference to locate the desired fragment. (3) When recovering DNA, excessive centrifugation should be avoided to prevent DNA from being sheared and to reduce library quality. Third, the choice of carrier At present, commonly used genomic library vectors include a cosmid vector, a P1 phage vector, a PAC vector (P1 artificial chromosome), a BAC vector (bacterial artificial chromosome), and a YAC vector (yeast artificial chromosome). Which carrier to choose can refer to the following factors: 1. Target area size If the target region of the genome is small (less than 50 kb), then a cosmid vector or even a lambda phage vector can be selected. Using existing commercial cosmid vectors, highly efficient packaging mixtures, and suitable E. coli strains, researchers can easily construct a cosmid vector library. If the target area of ​​the genome is large, then P1, PAC or BAC is more appropriate. P1 operation is difficult, and PAC is relatively simple. BAC vectors are also a good choice, and researchers can use existing mature products to build BAC libraries, such as a range of BAC vectors and kits from Biochem. If the target area is very large (greater than 250kb), the YAC carrier is preferred. However, the use of YAC vectors to construct genomic libraries is very cumbersome and difficult to perform, usually by a specialized company. Table 1 Comparison of various carriers Vector capacity (kb) host introduced into cell mode cosmid 30-45 E. coli transduction 2. The difficulty of screening libraries Cosmid vectors are typically screened using conventional filter-printing hybridization, but this method is laborious and wasteful for constructing regional maps and contigs. Large-capacity vector libraries, such as P1, PAC, BAC, and YAC, are stored in a two-dimensional array, either by conventional single-copy probe hybridization or by PCR for polyclonal population screening. Moreover, using a high volume vector to construct a library can reduce the steps of chromosome walking. 3. Consideration of carrier copy number: When a large fragment DNA fragment is cloned into a high-copy vector, the cloned DNA is recombined, thereby affecting the fidelity and stability of the cloned sequence; while the low-yield DNA of the single-copy vector is a bottleneck for high-throughput analysis. This contradiction often plagues researchers. The cloning system developed by Baibo Mingchuang over the years is the best solution to these difficulties. This system combines the advantages of single copy vectors with multiple copy vectors. The single copy vector can improve the stability of the insert, and the copy vector can immediately amplify a high copy number clone under the action of an inducer to obtain a high yield of DNA. 4. Link the genomic DNA fragment into the vector The above-described DNA fragment of the appropriate size is ligated into the vector using a ligase. Baibo Mingchuang has its own commercial carrier, which has been pretreated and can be used directly without endonuclease digestion and dephosphorylation. The ligase developed by Baibo Mingchuang has the advantages of fast connection and high efficiency. 5. Transfer the recombinant vector into the host cell If a cosmid library is constructed using a Biomaxon kit, the above-described ligation reaction product is first packaged, and the titer of the packaged cosmid clone is determined and then transfected. The recombinant is picked and the size of the insert is identified. If the size and quality of the library are satisfactory, the library can be screened, or the library can be expanded and saved. If the BAC library is constructed using the Baibo Mingchuang kit, the electroporation method should be selected. The Biocompressent E.coli can be selected as the host, and the above-mentioned ligation product is transferred into the bacteria, and the plate is grown and cloned. To obtain a clone, the investigator needs to evaluate the size of the BAC clone to determine if the library size is sufficient. The Baibo Mingchuang Library Construction Kit can easily identify the size of BAC clones and estimate the size of the BAC library. If the library is the right size, then the library can be used for subsequent operations. Determination of the number of library clones The genomic DNA library needs 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 the insert size to the size of the genomic DNA, and N is the number of clones required. For example, using the BAC vector to construct a human genomic library with a human genome size of 3 x 109 bp, an insert size of 100 kb, and a desired coverage of 99%, then the number of BAC clones required is N = ln (1 - 0.99) / Ln (1 - [105bp / 3 x 109 bp]) = -4.61 / -3.33 x 10-6 = 138,298 BAC Library Construction Screening Expert - Baibo Mingchuang Because of concentration, it is more professional! Baibo Mingchuang Company provides you with the most ideal library construction and screening technology services! Anesthesia and Breath Disposables Central Venous Catheter,Hemodialysis Catheter,Blood Pressure Transducer,Phripheral Inerted Central Catheter,Artery Compression Device Sampler,Infusion Pump Anesthesia Medical Co., Ltd. , https://www.jssinoanesthesias.com
Genomic DNA libraries have a wide range of uses, such as analysis and isolation of specific gene fragments for gene expression regulation, human and animal and plant genome engineering research. In general, the basic flow of genomic library construction can be classified into four major steps: isolating genomic DNA, performing related processing on genomic DNA, ligating genomic DNA fragments into vectors, and transferring recombinant vectors into host cells.
To construct a cosmid genomic DNA library, the researchers need to randomly splicing the genomic DNA with a syringe; then repairing the DNA with a terminal repair enzyme can increase the efficiency of DNA ligation into the vector; then find 40kb by pulsed field electrophoresis or common electrophoresis. The DNA fragments were left and right, and then DNA was recovered using a Biomines gel recovery kit. To construct a BAC genomic DNA library, the researchers need to digest genomic DNA with restriction endonucleases (usually EcoR I, BamH I or Hind III), and then find a DNA fragment of appropriate length by pulse field electrophoresis (eg 100kb-150kb). ), followed by dialysis to recover DNA.
P1 70-100 Escherichia coli transduction
PAC 130-150 E. coli
BAC 120-300 E. coli
YAC 250-400 Yeast Transformation