Anonymous
Feb 9
Outline five steps in construction of genomic library?
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Feb 9
1. Isolation of genomic DNA: The first step in constructing a genomic library is to isolate the genomic DNA from the organism of interest. This can be done using various methods such as cell lysis, proteinase K treatment, and phenol-chloroform extraction.
2. Fragmentation of genomic DNA: The isolated genomic DNA is then fragmented into smaller pieces. This can be achieved through mechanical shearing, enzymatic digestion, or sonication. The goal is to obtain a range of DNA fragments of different sizes.
3. Insertion of DNA fragments into a vector: The fragmented genomic DNA is ligated into a vector, which is typically a plasmid or a bacteriophage. The vector serves as a carrier for the DNA fragments and allows for their replication and expression in a host organism. The ligation is typically carried out using DNA ligase.
4. Transformation of host cells: The ligated vector containing the genomic DNA fragments is introduced into host cells, usually bacteria such as Escherichia coli. This is done through a process called transformation, where the host cells take up the vector DNA. The transformed cells are then grown on agar plates containing selective media to allow for the identification and isolation of cells that have taken up the vector.
5. Screening and identification of clones: Once the transformed cells have been grown, the genomic library can be screened to identify specific clones of interest. This can be done using various methods such as colony hybridization, polymerase chain reaction (PCR), or DNA sequencing. The identified clones can then be further analyzed and studied for specific genes or sequences of interest.
2. Fragmentation of genomic DNA: The isolated genomic DNA is then fragmented into smaller pieces. This can be achieved through mechanical shearing, enzymatic digestion, or sonication. The goal is to obtain a range of DNA fragments of different sizes.
3. Insertion of DNA fragments into a vector: The fragmented genomic DNA is ligated into a vector, which is typically a plasmid or a bacteriophage. The vector serves as a carrier for the DNA fragments and allows for their replication and expression in a host organism. The ligation is typically carried out using DNA ligase.
4. Transformation of host cells: The ligated vector containing the genomic DNA fragments is introduced into host cells, usually bacteria such as Escherichia coli. This is done through a process called transformation, where the host cells take up the vector DNA. The transformed cells are then grown on agar plates containing selective media to allow for the identification and isolation of cells that have taken up the vector.
5. Screening and identification of clones: Once the transformed cells have been grown, the genomic library can be screened to identify specific clones of interest. This can be done using various methods such as colony hybridization, polymerase chain reaction (PCR), or DNA sequencing. The identified clones can then be further analyzed and studied for specific genes or sequences of interest.
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