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Appendix B
Application Notes
DNA/RNA Shield
™
Lysis Tubes (Microbe)
(Cat. No.
R1103
)
1. Collect sample directly into the
DNA/RNA Shield™ Lysis Tube (Microbe)
.
2. Directly proceed to Step 2 of the protocol (page 4) and bead beat in the DNA/RNA Shield™ Lysis Tube
(Microbe) according to provided recommendations.
3. Proceed with the remaining protocol as written.
DNA Viruses
For unbiased metagenomics analysis of viruses, incorporating a Proteinase K digestion prior to bead beating is
recommended.
1. Following Step 2 (page 4) add 5% (v/v) of
Proteinase K
(Cat. No. D3001-2-5) to the lysate within the
ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm)
and incubate for 30 minutes at 55˚C.
2. Proceed to Step 3 (page 4) and continue with the remaining protocol as written.
Cheese and Protein Rich Biofluids (e.g. Milk, Sputum, Saliva, Spinal Fluid, Blood, and Serum)
1. Add ≤ 0.4 g of cheese or ≤ 200 µl of biofluid to the
ZR BashingBead
™
Lysis Tubes (0.1 & 0.5 mm)
.
Add 750 µl of
ZymoBIOMICS
™
Lysis Solution
.
2. Add 20 µl of
Proteinase K
(20 µg/µl) (cat. no. D3001-2-5) to the ZymoBIOMICS™ Lysis Tubes (0.1 & 0.5 mm)
and incubate for 30 minutes at 55˚C.
3. Continue to Step 2 (page 4) and proceed with the protocol as written.
Plant Tissue
(Leaves and other plant material)
Plant tissues such as leaves and roots contain DNA sources within the host tissue that can overwhelm 16S rRNA
gene targeted sequencing (from both mitochondria & chloroplast). Microbes must be removed from the plant
surface to exclude host tissue from the bead beating process.
(A)
Plant tissue – Centrifugation of cells
1. Suspend plant tissue in isotonic solution (
e.g.
PBS) and gently sonicate or vortex briefly.
2. Remove plant tissue from solution and centrifuge at 15,000 x g for 10 minutes to pellet the cells.
3. Without disturbing the pellet, slowly decant or pipette out the supernatant, leaving behind 100 – 300 µl of pellet.
4. Add
ZymoBIOMICS™ Lysis Solution
to the cells to a final volume of 1 ml and mix to resuspend. Transfer the
mixture to the
ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm)
and proceed to Step 2 (page 4).
(B)
Plant tissue – Filtration of cells
1. Place plant tissue in a submerging volume of PBS inside of a conical tube and gently sonicate or vortex briefly.
Remove plant tissue from liquid volume.
2. Filter liquid using a 0.22 µm filter (not provided).
3. Cut the filter and place directly into the
ZR BashingBead
™
Lysis Tubes (0.1 & 0.5 mm)
and proceed to
Step 1 (page 4).
