Introduction
Genomic DNA is frequently isolated from animal fecal samples to detect bacterial pathogens. Often DNA isolated from these sample types will contain significant amounts of inhibitors, such as phenolics and polysaccharides, which may prevent DNA amplification. In many cases, it may be necessary to dilute the purified DNA or treat with Chelex® resin or PVPP (polyvinylpolypyrrolydone) for successful amplification. Using the Maxwell® 16 Tissue DNA Purification Kit, bacterial DNA from mouse fecal samples was successfully isolated and the ability to amplify the purified DNA was achieved.
Methods
Genomic DNA Isolation: Fecal pellets collected from three-day old mouse bedding were stored at –20 °C until use. A single fecal pellet (average weight = 38mg) was placed directly into the Maxwell® 16 Tissue DNA Purification Kit (Cat.# AS1030) sample cartridge. Genomic DNA was purified using the Maxwell® 16 Instrument (Cat.# AS1000; firmware version 3.2, NOTE: Cat.# AS2000 now replaces Cat.# AS1000) as described in the Maxwell® 16 DNA Purification Kits Technical Manual #TM284. For some experiments, the eluted DNA was transferred to a prepared Zymo-Spin™ IV-HRC Spin Filter (Zymo Research Cat.# C1010-50) and centrifuged at 8,000 × g for 1 minute per the manufacturer’s instructions. DNA yield and purity were determined using the NanoDrop® spectrophotometer.
PCR Analysis: PCR was performed on aliquots of DNA from a minimum of six separate purifications using conserved eubacterial 16S ribosomal RNA primers (1)
. E. coli JM109 genomic DNA [isolated from 0.4ml overnight culture using the Maxwell® 16 Cell DNA Purification Kit (Cat.# AS1020) was used as a positive control. One microliter of eluted DNA or DNA diluted 1:100 in water was used per 50µl amplification reaction.
Results
Using single fecal pellets, we obtained an average of 48µg DNA per sample (n = 8). The DNA had an average concentration of 308ng/µl with a 260nm/280nm absorbance purity ratio of 1.62.
As seen in Figure 1, the Maxwell® 16 Tissue DNA Purification Kit consistently isolated high-molecular-weight DNA from the fecal pellets. The mechanical action of the Maxwell® 16 Instrument plungers was sufficient to break apart the samples with minimal DNA shearing; so the conventional preprocessing method of bead beating was not necessary.
The ability to detect microbial flora in the fecal DNA was then tested using conserved eubacterial 16S ribosomal RNA primers (1; Figure 2). One out of 8 samples gave amplifiable product without dilution or further treatment of the purified DNA. When the DNA was diluted 1:100 in water prior to amplification, we were able to amplify DNA from all samples.
To improve amplification from undiluted DNA, we also tested a subsequent treatment of the Maxwell® 16 purified DNA that removes PCR inhibitors. After pipetting the eluate onto a Zymo-Spin™ IV-HRC Spin Filter (filled with crosslinked polyvinylpyrrolidone) and centrifuging for 1 minute, the DNA could be used directly in PCR (Figure 2, lane 8; example is representative of six replicates).
Conclusion
The Maxwell® 16 System, which includes the Maxwell® 16 Tissue DNA Purification Kit and the Maxwell® 16 Instrument, can be used to isolate genomic DNA from mouse fecal pellets without any initial preprocessing steps. For subsequent PCR amplification, dilution of the purified DNA or removal of inhibitors over a Zymo-Spin™ IV-HRC Spin Filter is recommended for greatest success. To learn more about the Maxwell® 16 System, visit www.promega.com/maxwell16.