Product Application
DNA Purification from OMNIgene®•VAGINAL microbiome collectiondevices
Purify bacterial DNA from OMNIgene®•VAGINAL microbiome collection devices using the Maxwell® RSCPureFood GMO and Authentication Kit.
Kit:Maxwell® RSC PureFood GMO and Authentication Kit (Cat.# AS1600)

Analyses:

NanoDrop™, QuantiFluor® ONE dsDNA, qPCR, NGS

Sample Type(s):OMNIgene®•VAGINAL microbiome collection devices(Cat. #OMR-130 from DNA Genotek1)

Input:

600µl

Materials Required: Heat block Centrifuge
 Casework Spin Basket (Cat. #AS8101)
 Casework Microfuge Tube (Cat. #AS8201)
 Bead-beating tube (ex: MP Biomedicals Lysing Matrix SS) Bead-beater (ex: MP Biomedicals FastPrep-24™ 5G Instrument) Maxwell® RSC Instrument (Cat.# AS4500)

This protocol was developed by
Promega Applications Scientists andis intended for research use only.
Users are responsible fordetermining suitability of the
protocol for their application.
For further information, seeTechnical Manual TM473,
available at:www.promega.com/protocols
or contact Technical Services at:techserv@promega.com

Protocol:1. Add 40µl of Proteinase K and 20µl of RNase to swab and stabilization liquid. Invert tube 5-10times to mix.
2. Incubate lysate at 50°C for 1 hour.
3. Transfer swab into a Casework Spin Basket in a Casework Microcentrifuge Tube and centrifuge
at maximum speed for 2 minutes to remove all liquid from the swab.
4. Transfer all of the stabilization liquid to a 2ml Matrix SS tube (~1mL).
5. Bead-beat (Ex: 60 seconds using a QuickPrep Adapter on MP FastPrep-24™ 5G).
6. Add 600µl of lysate to the Maxwell® PureFood GMO and Authentication Kit cartridge and follow
TM473 Section 5 Purifying DNA on the Maxwell® Instruments.
a. Add 300µl of Lysis Buffer to well #1 of each cartridge.
b. Place a Maxwell® RSC Plunger into well #8 of each cartridge.
c. Add 100µl of Elution Buffer to each Elution Tube.

PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330
SCIENTIFIC APPLICATIONS NOTE 03/20

Product Application
Results:
Samples were collected from six individuals with an OMNIgene®•VAGINAL microbiome collection
device. DNA was purified from 600µl of pre-processed lysate with the Maxwell® RSC PureFood GMO
and Authentication Kit as described above. NanoDrop™ purity ratios ≥ 1.7 suggest DNA eluates are highquality DNA (Figure 1) and 5 out of 6 samples yielded ≥ 1x108 16S rRNA gene copies/µl (Figure 2). Little
to no PCR inhibition was observed with 16S rRNA gene qPCR amplifications (data not shown).

Figure 1. NanoDrop™ and QuantiFluor® ONE dsDNA concentrations with NanoDrop™ purity ratios.
The left Y-axis is DNA concentration in ng/µl and the right Y-axis is the NanoDrop™ purity ratio.

Figure 2. Average 16S rRNA gene copies/µl of undiluted eluates. N=2 amplification replicates using 2µlof undiluted eluate. Y-axis is log scale.

PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330
SCIENTIFIC APPLICATIONS NOTE 03/20

Product Application
Eluates were analyzed with Loop Genomics LoopSeq™ 16S Long Read Kit to sequence full-length 16S
rRNA genes. The library preparation was performed as indicated by the manufacturer, except for threemodifications (1) sample concentration was measured by the QuantiFluor® ONE dsDNA System, (2) all
clean-up steps in the workflow were performed with the ProNex® Size-Selective Purification System, and(3) library concentration was measured using the ProNex® NGS Library Quant Kit. Libraries weresequenced with 2x300 reads on an Illumina MiSeq instrument. Data was analyzed with the Loop
Genomics bioinformatics workflow.
The organisms detected were grouped by taxonomy which were then used to calculate the relative
abundance of bacterial taxa in the population. As expected with vaginal samples, the population isdominated by the Lactobacillus genus (≥ 96%); data not shown. Figure 3 shows the relative abundanceof genera other than Lactobacillus. With Lactobacillus excluded, donor specific differences are more
apparent in each sample.

Figure 3. Genus level comparison of bacterial profiles purified from vaginal swabs of 6 donors as
determined by LoopSeq™ 16S Long Read Kit with the genus Lactobacillus excluded.

OMNIgene®•VAGINAL is For Research Use Only, not for use in diagnostic procedures.
®OMNIgene is a registered trademark of DNA Genotek Inc.

1PROMEGA CORPORATION • 2800 WOODS HOLLOW ROAD • MADISON, WI 53711-5399 USA • TELEPHONE 608-274-4330
SCIENTIFIC APPLICATIONS NOTE 03/20