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ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Protocol
1. Add sample to a
ZR BashingBead
™
Lysis Tubes (0.1 & 0.5 mm)
. Add
750 µl
ZymoBIOMICS
™
Lysis Solution
to the tube and cap tightly.
Note:
For samples stored and lysed in
DNA/RNA Shield™ Lysis Tubes
, do not add
ZymoBIOMICS™ Lysis Solution and proceed to Step 2.
Sample Type
Maximum Input
Feces
200 mg
Soil
250 mg
Liquid Samples
1
and Swab Collections
2
250 µl
Cells (isotonic buffer,
e.g.
PBS)
50-100 mg (wet weight)
(10
9
bacterial and 10
8
yeast cells)
Samples in DNA/RNA Shield
™,3
≤ 1 ml
2. Secure in a bead beater fitted with a 2 ml tube holder assembly and
process at maximum speed for ≥ 5 minutes.
Note:
Processing time will vary based on sample input and bead beater. Times
may be as little as 5 minutes when using high-speed cell disrupters (FastPrep
®
-24) or as long as 20 minutes when using lower speeds (e.g., Disruptor Genie
â
).
4
3. Centrifuge the
ZR BashingBead
™
Lysis Tubes (0.1 & 0.5 mm)
in a
microcentrifuge at ≥ 10,000 x
g
for 1 minute.
4. Transfer up to 400 µl supernatant to the
Zymo-Spin™ III-F Filter
in a
Collection Tube
and centrifuge at 8,000 x
g
for 1 minute. Discard the
Zymo-Spin™ III-F Filter.
5. Binding preparation:
Feces and All Non-Soil Samples
OR
Soil Samples
Add 1,200 μl of
ZymoBIOMICS
™
DNA
Binding Buffer
to the filtrate in the
Collection Tube from Step 4. Mix well.
Add 800 μl of
ZymoBIOMICS
™
DNA
Binding Buffer
and 400 μl of 95%
ethanol
to the filtrate in the Collection
Tube from Step 4. Mix well.
6. Transfer 800 µl of the mixture from Step 5 to a
Zymo-Spin
™
IICR Column
in a Collection Tube and centrifuge at 10,000 x
g
for 1 minute.
7. Discard the flow through from the Collection Tube and repeat Step 6.
8. Add 400 µl
ZymoBIOMICS
™
DNA Wash Buffer 1
to the Zymo-Spin
™
IICR Column in a new Collection Tube and centrifuge at 10,000 x
g
for
1 minute. Discard the flow-through.
9. Add 700 µl
ZymoBIOMICS
™
DNA Wash Buffer 2
to the Zymo-Spin
™
IICR Column in a Collection Tube and centrifuge at 10,000 x
g
for
1 minute. Discard the flow-through.
For
Technical Assistance
:
1-888-882-9682 or E-mail
[email protected]
1
For water samples, filter
using desired filter (not
provided). Cut the filter into
small pieces and place into
ZR BashingBead
™
Lysis
Tubes (0.1 & 0.5 mm).
2
Swabs can also be cut or
broken, then placed directly
in bead beating tube. For
more information on
processing swab samples,
see Appendix B.
3
Up to 1 ml of sample in
DNA/RNA Shield can be
processed directly in ZR
BashingBead™ Lysis Tube.
Adjust final volume to 1 ml
with ZymoBIOMICS™ Lysis
Solution or DNA/RNA
Shield, if necessary.
4
For optimal lysis efficiency
and unbiased profiling, all
bead beater devices beyond
those validated by Zymo
Research should be
calibrated using the
ZymoBIOMICS™ Microbial
Community Standard (see
Appendix C).
