Rt pcr reagents

The cloning method was adapted from a protocol published by Smith et al (2009)(13 (link)). Briefly, 96-well PCR plates containing sorted plasmablast were thawed and used directly for reverse transcription PCR by the addition of dNTPs, RT-PCR buffer, RNAase-free H₂O, reverse transcriptase, and two primer pools containing forward and reverse primers for the heavy and light chains (RT-PCR reagents purchased from Qiagen, primers purchased from Geneart, Invitrogen). These PCR products were used in 3 separate nested PCR reactions to amplify the heavy and light chains separately, and to introduce restriction sites to the ends of the PCR products. Next the PCR products were digested using their respective restriction enzymes (AgeI-HF, SalI, XhoI, and BsiWI, all purchased from New England Biolabs) and ligated into human IgH, IgK, and IgL vectors containing a CMV promotor and ampicillin resistance (all expression vectors were kindly provided by Dr Juthathip Mongkolsapaya) using T4 DNA ligase (New England Biolabs). Vectors were expanded using TOP10 chemically competent E. Coli (Invitrogen), purified using QIAprep Spin Miniprep Kit (Qiagen), and transfected into HEK293/T17 cells in FreeStyle 293 expression medium (Thermo Scientific). Antibody supernatants were collected after 2 days of culture and stored at 4°C for further use.