Sanger sequencing and cloning of BTKCys481 mutants

A 382 bp fragment covering the BTK^Cys481 was amplified by PCR. The forward and reverse PCR primers were 5′-TGAGAAGCTGGTGCAGTTGTATG-3′ and 5′-CTGGAGATATTTGATGGGCTCAG-3′, respectively. The amplified PCR products were isolated by QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), and sequenced using forward and reverse PCR primers. PCR products were cloned into the TA cloning vector, and selected colonies sequenced using the M13 primers (Genewiz, South Plainfield, NJ).