ZYMO RESEARCH CORP.
Phone: (949) 679-
1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com
Page 6
Centrifugation Protocol: (Alternative)
Perform steps 1-8 as indicated in the general protocol, see page 4.
9. Place a
Zymo-Spin
™
II-P
Column
in a Collection Tube and transfer the entire
mixture from step 8 into the Zymo-Spin
™
II-P Column.
10. Incubate the
Zymo-Spin
™
II-P/Collection Tube
assembly at room temperature for
2 minutes and then centrifuge at 5,000
x g
for 1 min. Discard the flow through
1
.
11. Add 800
µ
l of
ZymoPURE
™
Wash 1
to the Zymo-Spin
™
II-P Column
and centrifuge
at 5,000
x g
for 1 min. Discard the flow through.
12. Add 800 µl of
ZymoPURE
™
Wash 2
to the Zymo-Spin
™
II-P Column and centrifuge
at 5,000
x g
for 1 min. Discard the flow through.
13. Add 200 µl of
ZymoPURE
™
Wash 2
to the Zymo-Spin
™
II-P Column and centrifuge
at 5,000
x g
for 1 min. Discard the flow through.
14. Centrifuge the Zymo-Spin
™
II-P Column
at ≥ 10,000 x
g
for 1 minute in order to
remove any residual wash buffer.
15. Transfer the Zymo-Spin
™
II-P Column into a clean 1.5 ml tube and add 25 µl of
ZymoPURE
™
Elution Buffer
2,3
directly to the column matrix. Incubate at room
temperature for 2 minutes, and then centrifuge
at ≥ 10,000 x
g
for 1 minute in a
microcentrifuge
. Store the eluted plasmid DNA at ≤ -20°C.
Notes:
1
The capacity of the
collection tube with the
column inserted is 900 µl.
Empty the collection tube
whenever necessary to
prevent contamination of the
spin-column with the flow
through.
2
The
ZymoPURE
™
Elution
Buffer
contains 10 mM Tris-
HCl, pH 8.5, 0.1 mM EDTA.
If required, pure water can
also be used to elute the
DNA.
3
The DNA yield can be
increased by pre-warming the
Zymo PURE
™
Elution Buffer
to 50 ºC and/or increasing the
incubation period up to 5
minutes prior to centrifugation.