Protein Engineering Protocols - Mycobacteriology research center

170 Stebel et al.9. Gel extraction kit (GE Healthcare or Qiagen).10. PCR cleanup kit (GE Healthcare or Qiagen).2.3. Enzymatic Digest and Chemical Cleavage1. uracil–DNA–glycosylase (UDG) from E. coli (NEB or Peqlab BiotechnologieGmbH).2. 99.9% piperidine (Sigma).2.4. Denaturing Polyacrylamide Urea Gels1. Urea (purity, at least 99.5%).2. 30% polyacrylamide/0.8% bis-acrylamide (37.5:1) stock solution (Carl Roth GmbH).3. 10X TBE buffer (see Subheading 2.2.6.).4. 10% ammoniumperoxodisulfate in water.5. TEMED.6. Deionized formamide.7. Defined oligonucleotides serving as marker or a low-range DNA ladder, e.g., a100-basepair (bp) ladder (NEB).8. 6X bromphenol blue loading buffer: 0.25% (w/v) in 70% (w/v) sucrose solution.9. Ethidium bromide solution: 5 mg/mL in water.2.5. Fragment Purification2.5.1. Direct Purification From Piperidine Cleavage1. QiaexII kit (Qiagen).2. Acetate buffer: 3 M Na-acetate, pH 5.3.3. Elution buffer: 10 mM Tris-HCl, pH 8.0.2.5.2. Purification From Denaturing Polyacrylamide Urea Gels1. Qiaex II purification: diffusion buffer: 0.5 M ammonium acetate, 10 mM magnesiumacetate, 1 mM EDTA, and 0.1% sodium dodecyl sulfate, pH 8.0.2. Water extraction: deionized sterile water.3. Acetate buffer: 3 M Na-acetate, pH 5.3.4. 1 M MgCl 2.5. 2-Propanol.6. 90% ethanol.7. Elution buffer: 10 mM Tris-HCl, pH 8.0.2.6. Quantification of Purified Fragments1. 1:5000 diluted SYBR green II (Molecular Probes).2.7. Gene Reassembly and Amplification1. 10 mM mixtures of dATP, dTTP, dCTP, and dGTP.2. Vent DNA Polymerase (NEB).3. 25 mM MgSO 4.4. Taq DNA polymerase (from different suppliers).