The MasterPure™ Gram-Positive DNA Purification Kit provides all of the reagents needed to purify DNA from Gram-positive bacteria. These bacteria lyse more readily after treatment with Ready-Lyse™ Lysozyme and the Gram-Positive Cell Lysis Solution included in the kit. Ready-Lyse Lysozyme is a stable solution of a recombinant lysozyme from a nonmammalian, nonavian source. It has high specific activity and does not bind DNA.1,2
Examples of Gram-positive bacteria tested with this kit are listed in Table 1. The size range of DNA purified by the MasterPure Gram-Positive DNA Purification Kit is shown in Fig. 1. The DNA purified using the kit is suitable for PCR analysis (Fig. 2), and other molecular biology applications.
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Incubation Times Needed for DNA Recovery from 1ml of Gram Positive Bacterial Culture
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Species
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Culture Medium
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Ready-Lyse™ Incubation
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DNA Yield µg/ml
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Bacillus subtilis
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Brain-Heart Infusion (BHI)
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30 min
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9.0
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Listeria monocytogenes
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BHI
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Overnight
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3.3
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Staphylococcus aureus
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BHI
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Not needed
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8.0
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Streptococcus mutans
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Todd-Hewitt
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Overnight
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3.0
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Lactococcus lactis
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M17
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30 min
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1.1
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Figure 1
B. subtilis DNA |
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Figure 2![Figure 2. PCR Product from DNA Purified Using the MasterPure™ Gram Positive DNA Purification Kit. B. subtilis DNA (1ul containing 0.5 ng) was amplified by PCR using forward primer 5'-AGG GTC ATT GGA AAC TGG G and reverse primer 5'-CGT GTT GTA GCC CAG GTC ATA. The cycle conditions were 95°C for 2 min and then 30 cycles of [95°C for 45 sec; 55°C for 45 sec; 72°C for 45 sec] followed by 72°C for 2 min. Lane 1, 100 bp ladder. Lane 2, PCR product from 0.5 ng of B. subtilis DNA. Lane 3, no template PCR](/library/images/html_images/masterpure_grampos_fig2.gif)
PCR of BacF/BacR |
Figure 1. Electrophoretic Analysis of DNA Purified Using the MasterPure™ Gram Positive DNA Purification Kit. A. DNA from Bacillus subtilis (ATCC 6051) was separated on a 1% agarose gel and stained with SYBR® Gold.Lane 1, kilobase ladder. Lane 2, 300 ng of B. subtilis DNA. B. Pulsed field gel electrophoresis of Bacillus subtilis (ATCC 6051) DNA. Lane 1, Phage lambda ladder. Lane 2, 300 ng of DNA. Lane 3, 600ng of DNA.
Figure 2. PCR Product from DNA Purified Using the MasterPure™ Gram Positive DNA Purification Kit. B. subtilis DNA (1ul containing 0.5 ng) was amplified by PCR using forward primer 5'-AGG GTC ATT GGA AAC TGG G and reverse primer 5'-CGT GTT GTA GCC CAG GTC ATA. The cycle conditions were 95°C for 2 min and then 30 cycles of [95°C for 45 sec; 55°C for 45 sec; 72°C for 45 sec] followed by 72°C for 2 min. Lane 1, 100 bp ladder. Lane 2, PCR product from 0.5 ng of B. subtilis DNA. Lane 3, no template PCR.
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