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simultaneous mutagenesis of two or more contiguous amino acid positions is done using an oligonucleotide that contains contiguous N,N,G/T triplets, i.e

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The filtered supernatant was run over a protein A/G column (Pierce), followed by extensive washing with PBS and the antibodies were eluted with 18 mL 0.1 M glycine pH 2.5 directly captured in 2 mL neutralization buffer (1 M TRIS pH 8.7)