A rapid and inexpensive one-tube genomic DNA extraction method from Agrobacterium tumefaciens

Abstract

Many methods have been used to isolate genomic DNA, but some of them are time-consuming and costly, especially when extracting a large number of samples. Here we described an easy protocol using two simple solutions for DNA extraction from A. tumefaciens cells. Compared with the standard protocol, this protocol allows rapid DNA isolation with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) gDNA extraction was achieved within 15 min; (2) this method was cost-effective, since it only used calcium chloride and lysozyme; SDS, phenol, chloroform and proteinase K were not necessary; (3) the method gave high yield of gDNA (130 ng/loopful culture) compared with standard protocol that was suitable for restriction analysis; (4) the protocol can be carried out in a single test tube and the cells directly from solid media can be used. Thus, this protocol offers an easy, efficient and economical way to extract genomic DNA from A. tumefaciens.

Begomovirus detection in the whitefly Bemisia spp. on eggplant Solanum melongena L. leaves

“…using lysis buffer containing 0.01 M Tris-HCl pH 8.0, 0.01 M NaCl, 0.005 M CaCl 2 , 0.002 M EDTA pH 8.0, 2% SDS, β-mercaptoethanol, and 0.25 mg/mL of the Proteinase-K. Cell lysis was performed using micro pestle, while lysis buffer containing CaCl 2 and SDS played a role in weakening cell membranes as well as dissolving labile fragments [21,22] . Furthermore, Tris and EDTA pH 8.0 maintained pH stability and bind ions in charge of maintaining the integrity of cell membranes [23,24].…”

“…Tris and EDTA were used to stabilize pH and assist the process of membrane lysis, as well as DNase inactivation [21,22]. The removal of RNA, protein, carbohydrates, tannins, polyphenols, lipids contamination from DNA was assisted by RNase, β-mercaptoethanol, Proteinase-K, PVP, phenol, and chloroform [24].…”