Product Application
DNA Purification from OMNIgene®•VAGINAL microbiome collectiondevices
Purify bacterial DNA from OMNIgene®•VAGINAL microbiome collection devices using the ReliaPrep™
Blood gDNA MiniPrep System.
Kit:

ReliaPrep™ Blood gDNA MiniPrep System (Cat. #A5081)

Analyses:

NanoDrop™, QuantiFluor® ONE dsDNA, qPCR, NGS

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

Input:

300µl

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)This protocol was developed by
Promega Applications Scientists andis intended for research use only.
Users are responsible fordetermining suitability of theprotocol for their application.
For further information, seeTechnical Manual TM330,
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 (optional) to swab and stabilization liquid. Invert
tube 5-10 times to mix.
2. Incubate 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 MP FastPrep-24™ 5G).
6. Add 300µl of lysate to a clean 1.5mL tube.
7. Add 600µl of 100% Isopropanol and gently invert to mix.
8. Add 300µl of Cell Lysis Buffer to lysate. Gently mix by inversion.
9. Load 600μl of sample into a ReliaPrep™ Binding Column placed in a collection tube, spin for 1
minute at maximum speed. Discard flow through.
10. Load the rest of the sample on to the ReliaPrep™ Binding Column, and spin for 1 minute atmaximum speed. Place Binding Column into a new collection tube.
11. Add 500μl of Column Wash Buffer (CWD).
12. Spin 2 minutes at maximum speed and then discard flow through.
13. Repeat steps 11 and 12 twice, for a total of three washes.
14. Place Binding Column in a labeled elution tube. Add 100µl of pre-warmed (50°C) Elution Buffer
to Binding Column. Spin 1 minute at maximum speed.

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 300µl of pre-processed lysate with the ReliaPrep™ Blood gDNA MiniPrep
System as described above. NanoDrop™ A260/A280 purity ratios were ≥ 1.7 while A260/A230 ratios
were near 1.0 (Figure 1). Samples yielded an average of 9.4x107 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) allclean-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 relativeabundance of bacterial taxa in the population. As expected with vaginal samples, the population isdominated by the Lactobacillus genus (≥ 93%); data not shown. Figure 3 shows the relative abundanceof genera other than Lactobacillus. With Lactobacillus excluded, donor specific difference are more
apparent in each sample.

Figure 3. Genus level comparison of the 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.

1

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