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MasterPure™ Gram Positive DNA Purification Kit - Frequently Asked Questions

The MasterPure Gram Positive DNA is designed for rapid, high-yield purification of DNA from Gram positive bacterial species. The kit includes Ready-Lyse Lysozyme, a recombinant enzyme with high specific activity that facilitates lysis of Gram positive bacteria. The procedure provides high yields of pure, intact bacterial DNA without bead-beating, spin columns, or toxic chemicals (Figure 1).


Figure 1. The MasterPure Gram Positive DNA Purification Kit provides high yields of pure total DNA.
The kits can be used with a variety of bacteria, including Bacillus subtilis, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, and many other species.
The purified bacterial DNA is suitable for a variety of molecular biology applications, such as cloning, PCR amplification, Southern blotting, genomic library preparation, and next gen sequencing (NGS). Researchers have published many novel applications for the MasterPure Gram Positive DNA Purification Kit, including: quality validation of commercial probiotics by NGS [1]; NGS-based mechanistic studies of multidrug-resistant Klebsiella pneumonia [2]; identification of a novel bacterial species in ground beef by capillary sequencing [3]; and developing a model system to study periodontal disease using qPCR in a multispecies biofilm [4]. In addition, the kit has been to purify DNA for obtaining draft and complete genomes of a range of bacterial strains and serotypes, including two Propionibacterium acnes strains in patients with skin diseases [5].
Due to the relatively thick cell wall and peptidoglycan layer, Gram positive bacteria can be difficult to lyse by ordinary methods. The MasterPure Gram Positive Kit procedure includes a special lysis step, using Ready-Lyse Lysozyme that is performed for 30 minutes to overnight, depending on the species, before protein removal and DNA precipitation. However, the kit will also work with Gram negative bacteria, so you can use the same kit for both types of bacteria.
The kit protocol can be scaled proportionally, depending on the volume of liquid culture harvested and the growth conditions. The protocol provided in the kit is based on starting with 1 mL of an overnight Gram Positive bacterial culture. The final yield of DNA will vary, depending on the bacterial species. Typical DNA yields when starting with a 1 mL overnight culture include 9 µg for Bacillus subtilis, 3 µg for Streptococcus mutans and Listeria monocytogenes, 4 µg for Staphylococcus epidermidis, and 8 µg for Staphylococcus aureus.
Yes, the kit has been used successfully to purify DNA for both NGS and capillary sequencing.
Spectrophotometric methods (e.g., A260) to quantify DNA or RNA, although in common use, may overestimate the concentration. The best method to quantify DNA is by fluorometry using a DNA-specific dye, such as Hoechst 332581 (bisbenzimide), or PicoGreen® dye (Thermo Fisher Scientific). These dyes bind specifically to double-stranded DNA and not to nucleotides, single-stranded DNA, or RNA.

References

  1. Lewis ZT et al. 2016. Validating bifidobacterial species and subspecies identity in commercial probiotic products. Pediatr Res 79:445-452.
  2. Jana B et al. 2017. The secondary resistome of multidrug-resistant Klebsiella pneumoniae. Sci Rep 7:42483
  3. Rooney AP et al. 2011. Peptoniphilus methioninivorax sp. nov., a Gram Positive anaerobic coccus isolated from retail ground beef. Intl J Syst Evol Microbiol 61:1962-1967.
  4. Millhouse E et al. 2014. Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules. BMC Oral Health 14:80.
  5. Petersen R et al. 2015. Draft genome sequences of two Propionibacterium acnes strains isolated from progressive macular hypomelanosis lesions of human skin. Genome Announc 3:e01250-15.

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