The mainstream genome editing tools include clustered regularly interspaced short palindromic repeat (CRISPR)/CAS9 RNA-guided nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and helper dependent adenoviral vectors (HDAdVs). The process and protocols of mainstream genome editing methods from literature: 1). the CRISPR-Cas9 system: – Genome engineering of mammalian haploid embryonic stem […]
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The general hybridoma antibody cloning protocol and antibody sequencing protocol seem very straightforward: RNA extraction, reverse transcription to cDNA, nested PCR amplifications, cloning of PCR products, sequencing, and analysis of sequences. However, it has taken scientists a long time to optimize a variety of parameters in the protocols, especially V-gene specific primers and antibody sequence […]
Similar to process of hybridoma or clonal B cell antibody cloning and sequencing, the work flow of single B cell antibody cloning and sequencing is: separation of peripheral blood mononuclear cells (PBMC) from blood, staining of PBMC with the B cell selective marker (CD19 antibody), sorting of stained B cells by flow cytometry, total RNA […]
Two mainstream strategies have been used to sequence antibody repertoires. The classical strategy is to design primers to cover as many functional V-genes as possible and create a diverse antibody libraries. The modern strategy is to use high-throughput next generation sequencing (NGS) to “deeply” sequencing of binding populations. The abundance of antibody mRNA can also […]
The key for successful scFv phage library construction is efficient two-step PCR amplification of heavy chain (VH) and ligh chain (VL) variable regions. scFv phage library construction and scFv phage library screening protocols from public literatures: – Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library. […]
Three mainstream strategies have been used to clone and subclone PCR-amplified DNA fragments. The traditional method is to digest the PCR products with restriction enzymes and ligate the digested DNA fragments with a digested vector. The second ligation strategy is called “TA cloning” based on the feature of ends of PCR products. The modern cloning […]