Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 2: MATERIALS & METHODSSection 2.3: Molecular Biology Methodologies2.3.1 Extraction of Bacterial DNAThe total genomic DNA content was extracted from isolated cultures following amodified published protocol (Rainey et al., 1992). An additional final purificationstep was incorporated into the protocol to ensure purity of the DNA.The processing of the cultured isolates for DNA extraction involved scrapingactinobacterial mycelium from the surface of a 10 day-old colony on yeast extractmaltextract agar (2 loopfuls) and incubating the culture in 400 μl saline-EDTAextraction buffer (0.15M NaCl, 0.1M EDTA, pH 8) with added lysozyme (10 mg/ml)(Sigma Chemical Co. St. Louis, MO., Cat. No. L7651) at 37 o C and then digesting thebacterial cells with two proteolytic enzymes, lysozyme and 1 % (w/v) proteinase K(Sigma Chemical Co. St. Louis, MO., Cat. No. P2308) in the presence of 25 % (w/v)SDS (Sigma Chemical Co. St. Louis, MO., Cat. No. L5750) and saline-EDTA buffer,causing lysis of the cell. Following incubation a phenol-chloroform extraction wasperformed followed by a ethanol precipitation step (Sambrook et al., 1989).Proceeding the ethanol precipitation of DNA a final purification step was carried outwith the Prep-A-Gene DNA Purification kit (BioRad, Hercules, CA., Cat. No. 732-6011). The DNA sample was loaded onto a ion-exchange resin where it bound, whilethe contaminants were washed out and the DNA was eluted off with water. Storage ofthe DNA extracts were kept at –20 o C.2.3.2 PCR Reaction ConditionsThe 5X reaction buffer consisted of 50 mM Tris HCI pH [8.3], 250 mM KCI, 7.5mMMgCI 2 and 0.2mM of each deoxynucleotide triphosphates (Boehringer andMannheim). Each PCR reaction was overlayed with 40 μl of mineral oil (SigmaChemical Co. St. Louis, MO., Cat. No. M-5904). The PCR was performed using aPerkin Elmer 480 Thermal Cycler (Perkin Elmer, Norwalk. USA)._____________________________________________________________________57