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What is the MasterPure Gram Positive DNA Purification Kit, and how does it work?

MGP04100, MB711400

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.

For what applications can I use the DNA purified with the MasterPure Gram Positive Kit?

MGP04100, MB711400

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; NGS-based mechanistic studies of multidrug-resistant Klebsiella pneumonia; identification of a novel bacterial species in ground beef by capillary sequencing; and developing a model system to study periodontal disease using qPCR in a multispecies biofilm. In addition, the kit has been used 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.

I work with both Gram-positive and Gram-negative bacteria. Can I use the MasterPure Gram Positive DNA Purification Kit for both types?

MGP04100, MB711400

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 DNA Purification 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.

How much starting material do I need for the MasterPure Gram Positive DNA Purification Kit? How much DNA will I get after purification?

MGP04100, MB711400

The kit protocol can be scaled proportionally, depending on the volume of liquid culture harvested and the growth conditions. The protocol provided with 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.

My AmpliScribe reaction had excellent yields, but when I ran it on a native gel the RNA was much shorter than the original template. What could have gone wrong?

AS3107, MBTOOL-007

Single stranded in vitro transcripts are best separated using denaturing gels1. Denaturing gels allow in vitro transcripts to separate on the basis of their length rather than based on their length + secondary structure. When using native gels, sample migration may be altered by secondary structures in the transcripts.

Do I need to perform the optional RNase treatment step in the MasterPure Yeast DNA Purification Kit protocol?

MPY03100, MPY80200

We recommend treating the cell lysate with RNase A (supplied in the kit) if the presence of small amounts of RNA will interfere with downstream applications, such as some PCR applications or sequencing library preparations. However, for applications such as restriction enzyme digestion and cloning, or PCR with primers specific to genomic DNA sequences, RNase treatment is not necessary.

Do I need to perform the optional DNase treatment step in the MasterPure Yeast RNA Purification Kit protocol?

MPY03100, MPY80200

The MasterPure Yeast RNA Purification Kit protocol recovers DNA with much lower efficiency than RNA after cell lysis, so removal of contaminating DNA may not be necessary for many applications. For applications such as RT-PCR with gene-specific primers, we recommend following the DNase treatment protocol.

Do you have data indicating concordance of Amp-Seq with other technologies?

AMPSEQ-001

Biosearch Technologies have internal data demonstrating concordance of Amp-Seq results with alternative technologies including Flex-Seq. We tested a panel of diverse samples with Flex-Seq and Amp-Seq with a concordance of 98.82%. If you would like more information about this, please contact techsupport@lgcgroup.com.

What is the best method for quantification of genomic DNA?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

We recommend using the Qubit® for quantification of DNA. However, high molecular weight genomic DNA is difficult to quantify, due to both its viscosity and the difficulty of accurately pipetting small volumes. We recommend re-quantification of your DNA after shearing, if possible, to get accurate information.

I’m seeing a lot of duplicates when I analyse my sequencing data generated on an Illumina sequencer with a patterned flow cell. Do you know the reason for these high duplication rates and is there a solution?

NXGSEQ-003, NXGSEQ-004, 15300-1, NXGSEQ-001, NXGSEQ-002

The majority of these duplicates are likely proximity duplicates produced during ExAmp cluster formation on patterned flow cells. These duplicates occur independently of the library and their severity may vary from run to run. They can be removed bioinformatically using a program such as Clumpify (a part of the BBMap toolset available at https://sourceforge.net/projects/bbmap/), which can remove proximity and optical duplicates from a dataset, as well as perform quality trimming and adaptor filtering.