A streamlined and reliable method to obtain high-quality single-stranded DNA
Jeff Braman Scott Basehore
The StrataPrep M13 DNA purification kit allows easy, quick, and reliable purification of bacteriophage single-stranded DNA. The yield of single-stranded DNA recovered from phage lysate exceeds the yield obtained with the leading commercial silica-gel-based (SGB) M13 DNA purification method. The resulting high-purity DNA is ideal for automated sequencing applications.
M13 DNA is a preferred template for many sequencing projects.1-5 For many years, the standard laboratory protocol used to purify M13 DNA relied upon polyethylene glycol (PEG)-NaCl precipitation of phage particles, phenol-chloroform extraction to remove phage coat proteins, and ethanol precipitation to concentrate the DNA.6 This method is time-consuming, employs hazardous chemicals, and is difficult to apply to more than a few samples.
However, with the StrataPrep M13 DNA purification kit, isolating high-purity M13 DNA is possible without these cumbersome steps because the procedure is streamlined: Collect M13 phage particles on the unique glass fiber matrix contained in a StrataPrep spin cup;7 add a chaotropic salt solution to the spin cup so the phage coat protein denatures and binds the liberated single-stranded DNA to the glass matrix; centrifuge-wash the phage DNA to remove contaminating protein and salt; and elute the DNA from the spin cup with a low ionic strength buffer. The concentration of the highly purified DNA that results is ideal for automated sequencing applications.
The yield of single-stranded DNA from StrataPrep M13 method of Phagescript lysate (a modified M13 bacteriophage8), was at least twice that obtained from the SGB method (Figure 1). For 3 ml of phage lysate, the StrataPrep method yielded 7.5 g of DNA at a concentration of 25 ng/l, as judged by gel electrophoresis.
Equal volumes of StrataPrep- and SGB-purified Phagescript DNA were subjected to fluorescent dye primer automated sequencing by an independent sequencing service (Sequetech Corp.; Figure 2). Signal intensity was more than twice as high for the StrataPrep-purified template (Figure 2, Panel A) compared to the SGB-purified template (Figure 2, Panel B). Sequence read length was nearly 700 bases for both templates. There were three and four sequence ambiguities scored for the StrataPrep- and SGB-purifed templates, respectively, within the first 20 nucleotides. Sequence ambiguities also occurred at nucleotides 553 and 638 for the StrataPrep-purified template and at nucleotide 44 for the SGB-purified template.
The StrataPrep M13 DNA purification kit contains a fast, easy, and reliable method for M13 purification. Recover single-stranded DNA from phage lysate more efficiently using the StrataPrep method than with the leading commercial silica-gel-based method, and realize high-quality automated fluorescent sequencing results as well. Additionally, the kit can be used to purify single-stranded DNA from rescued phagemid clones.