Peptide-Based Drug Design

164 Scocchi et al.
5. 50X Tris-Acetate-EDTA (TAE buffer): 2 M TRIS base, 5.2% (w/v) acetic acid
and 50 mM Na2-EDTA, pH 8.3.
6. 0.5 �g/mL ethidium bromide (Sigma) incorporated into the agarose gel.
7. T4 DNA Ligase 10 U/�L and its 10X reaction buffer (USB, GE Healthcare).
8. SOB medium: 20 g/L bacto-tryptone (Difco Laboratories), 5 g/L bacto-yeast
extract (Difco Laboratories), 0.5 g/L NaCl, 16.6 g/L KCl.
9. SOC medium: SOB medium with the addition of 20 mM sterile glucose and
10 mM sterile MgCl2.
10. Agar medium (see 2.1) containing 100 �g/mL ampicillin (ampicillin stock
solution: 100 mg/mL in MilliQ water, store at −20 ◦ C).
11. GFX TM Gel Purification Kit (GE Healthcare Biosciences).
12. Incubator set at 16 ◦ C.
13. Agarose gel electrophoresis apparatus.
14. Cuvettes for electroporation (Biorad).
15. Electroporation apparatus.
2.4. Identification of the Resistance Associated Genes
1. GFXTM Micro Plasmid Prep Kit (GE Healthcare).
2. EcoRI (10 U/�L), HindIII (10 U/�L), and their corresponding 10X reaction buffers
(Promega).
3. pUC18 universal primers M13 uni (−43) and M13 rev (−49).
2.5. Characterization of PRP Resistance
1. Buffered high-salt solution: 10 mM Na–phosphate, 400 mM NaCl, 10 mM MgCl2,
pH 7.5 (Buffer 3).
2. Agar medium, liquid broth (see Subheading 2.1).
2.6. Synthesis of Cys-Tagged and Fluorescently Labeled Peptides
1. Fluorophore: BODIPY � FL N-(2-aminoethyl)maleimide (Invitrogen); excitation
max at 505 nm, emission max at 513 nm. BODIPY � FL solution prepared fresh at
1 mg/ml in acetonitrile (AcCN).
2. Peptides with a cysteine residue added to the C-terminus to provide a free sulphydrile
group (for peptide synthesis protocols see ref. 15).
3. Phosphate-buffered saline (PBS).
4. Peptide stock solution 0.1 mg/mL in PBS containing 20% AcCN.
5. Analytical and semi-preparative reversed-phase columns (e.g. Waters Symmetry
and Delta-Pak TM C18; Millipore).
6. RP-HPLC and mass spectrometry instruments.
2.7. Flow Cytometry
1. Mueller-Hinton broth (see Subheading 2.1).
2. Buffer 3 (see Subheading 2.5).