Page 10
ZYMO RESEARCH CORP.
Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Bacterial Endospore Lysis
ZymoBIOMICS DNA Miniprep Kit is capable of effectively lysing bacterial endospores, and also achieves higher
yield when compared to competition.
Urine
(A)
Pelleting cells from fresh/frozen urine
1. Pellet the bacterial cells by centrifuging the urine at 15,000 x
g
for 10 minutes.
2. Without disturbing the pellet, slowly decant or pipette out the supernatant, leaving behind 100 – 400 µl of pellet.
3. Add
ZymoBIOMICS
™
Lysis Solution
to a final volume of 800 µl and then transfer the mixture to a
ZR BashingBead
™
Lysis Tube (0.1 & 0.5 mm)
. Proceed to Step 2 (page 4) and continue with the protocol as written.
(B)
Pelleting cells from stabilized urine
1. Add 70 µl
Urine Conditioning Buffer
(Cat. No. D3061-1-140) for every 1 ml of urine and mix well by vortexing.
Note:
Urine stabilized by the Urine Conditioning Buffer can be stored for up to 1 month at ambient temperature. When samples
are ready to be processed, mix well by vortexing, and proceed to Step 2.
2. Centrifuge at 3,000 x
g
for 15 minutes.
3. Without disturbing the pellet, slowly decant or pipette out the supernatant, leaving behind 100 – 400 µl of pellet.
4. Add
ZymoBIOMICS
™
Lysis Solution
to a final volume of 800 µl and then transfer the mixture to a
ZR BashingBead
™
Lysis Tube (0.1 & 0.5 mm)
. Proceed to Step 2 (page 4) and continue with the protocol as
written.
Figure 6.
DNA Extractions were performed using the ZymoBIOMICS
®
DNA Kit and DNeasy
PowerSoil with 6 x 10
8
B. subtilis
CFU
.
DNeasy PowerSoil reovered 0.62 ng/µl DNA, while the
ZymoBIOMICS
®
DNA Kit was capable of recovering 9.67 ng/
µ
l in a 50
µ
l elution volume.
Extractions were performed in triplicate and quantified via Qubit.
Figure 7.
Phylum composition of urine preserved in Urine
Conditioning buffer™ (UCB™), which preserves the microbial
composition of urine with simulated stool contamination for a
month at room temperature. Urine with UCB™ added (Zymo
Research, D3061-1-160) was stored at room temperature and
analyzed over a month period. At the indicated time points (0
Days, 2 weeks, and 1 month), DNA was extracted using the
ZymoBIOMICS
™
DNA Miniprep Kit. The extracted DNA was
then subjected to microbial composition profiling via 16S rRNA
gene targeted sequencing. Experiment was performed in
technical duplicates.
0 Day
2 Weeks 1 Month
