SMARTT: C. tetani glycine riboswitch (type-1) transcribed with 500 uM Gly - Preliminary Evaluation (Replicate B)
A mutagenized library of template DNA was made by error-prone PCR using the GeneMorph II Random Mutagenesis Kit (Agilent Technologies). Mutations were introduced to the aptamer and expression platform domains using a 16 cycle error-prone PCR reaction. Nonmutagenized regions upstream of the aptamer, including a promoter, were added through a two-step PCR reaction with Phusion HF polymerase (NEB or Thermo Fisher Scientific). This library was used as DNA template for an in vitro transcription reaction containing the specified concentration of glycine. DNA template was removed by DNase treatment with TURBO DNase (Thermo Fisher Scientific). A pre-adenylated adaptor was then added to the 3' end using T4 RNA Ligase II, Trunc. K227Q (NEB). Using this adaptor as a handle, the RNA was reverse-transcribed into DNA with SuperScript III (Thermo Fisher Scientific). Illumina adaptors were then added through a two-step PCR reaction with Phusion HF polymerase (NEB or Thermo Fisher Scientific).
SUB4500428
PE_Cte-02