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. Author manuscript; available in PMC: 2019 Jul 30.
Published in final edited form as: Methods Mol Biol. 2018;1764:101–121. doi: 10.1007/978-1-4939-7759-8_7

Fig. 2.

Fig. 2

Essential considerations needed for preparing site saturation mutagenesis (SSM) libraries. (a) The gene of interest is segmented in multiple libraries containing contiguous sections of 200–250 bp. Here, sections of 225 bp are shown for compatibility with 250 bp PE Illumina MiSeq sequencing. (b) Each mutagenic oligo contains an “NNK” codon to cover all possible 20 amino acids. (c) An Nt.BbvCI restriction enzyme (Nt) is used to create a nick on the sense strand. Mutagenic oligos are designed to be complementary to the antisense ssDNA template