L cysteine hydrochloride monohydrate

Isolation media consisting of Mycoplasma Broth Base (Becton Dickinson, Franklin Lakes, NJ) supplemented with dextrose (Thermo Fisher Scientific, Waltham, MA), L-cysteine hydrochloride monohydrate (Thermo Fisher Scientific), beta-NAD (Thermo Fisher Scientific), thallium acetate (Acros Organics, Fair Lawn, NJ), phenol red (Sigma Aldrich, St. Louis, MO), porcine serum (Quad Five, Ryegate, MT), equine serum (Quad Five), and penicillin G potassium salt (Sigma Aldrich) as both broth and agar plates. Mycoplasma plates were inoculated with the nasal wash used in the infection experiment. These plates were then incubated under microaerophilic conditions at 37°C for 10 days. Several of the resulting colonies were then determined to be M. ovipneumoniae through qPCR targeting the p133 gene (Yang et al., 2014 (link)) with Ct values <25. These colonies were inoculated into Mycoplasma broth and expanded by incubation under microaerophilic conditions at 37°C until acid production caused a color change in the media with the phenol red.

Oligonucleotides were purchased from Integrated DNA Technologies. T4 DNA ligase, T4 DNA ligase reaction buffer, T4 Polynucleotide Kinase (PNK), Q5 High-Fidelity DNA Polymerase (Q5), Q5 reaction buffer, Q5 High GC Enhancer, Deoxynucleotide (dNTP) Solution Mix, and DpnI were all purchased from New England Biolabs. E. coli strains NEB 5-α and Lemo21(DE3) were also purchased from New England Biolabs. Pierce Universal Nuclease for Cell Lysis and HisPur Cobalt Resin were purchased from Thermo Fisher Scientific. The French Press and the Manual-Fill 40K Cell (FA-032) were purchased from Glen-Mills. Q Sepharose Fast Flow was purchased from GE Healthcare. L-Selenocystine was purchased from Acros Organics. LCysteine hydrochloride monohydrate was purchased from Alfa Aesar.