This kit provides the specific primers requiredfor the amplification of mouse antibody variable regions (Gamma, Kappa, and IgG heavy chains) from hybridoma total RNA. The workflow involves:
Reverse Transcription: RNA is converted to cDNA using primers that bind conserved constant-region sequences. An adapter sequence is introduced during this step to provide a standardized priming site for subsequent amplification.
PCR Amplification: The resulting cDNA is amplified using a forward primer complementary to the adapter and reverse primers specific to the antibody chain type. A nested PCR design improves specificity and product yield.
Sequencing: The resulting ~500-600bp amplicons are purified for Sanger sequencing to determine the variable region DNA sequence.
Optimization: This kit is for sequencing variable regions of monoclonal IgG antibodies (subclasses IgG1, IgG2a, IgG2b, IgG2c) from hybridoma-derived mouse RNA, including both kappa and lambda light chains. It performs best with high-quality RNA (RIN > 7) from standard laboratory mouse strains and reliably produces ~500 to 600 bp amplicons using an adapter-based cDNA synthesis approach.
Limitations: Its primary limitations are poor amplification of mouse IgG3 due to primer mismatches, and it is not designed for non-IgG isotypes, non-mouse species, or heavily degraded RNA samples.
