••••••••••••••••••••••••
Spin Protocol
1.Place 1
175µl  R N A  L y s i s  B u f f e r  (R L A )(+ BME) in an autoclaved tube.2.Prepare sample for lysis.
3.Immediately place sample into L
L y s i s  B u f f e r . Mix thoroughly by inversion.N o t e :Ensure proper ratio of Lysis Buffer to sample. See Table 1 of the standard protocol.*
4.Add 3
350µl  R N A  D i l u t i o n  B u f f e r (RDA, blue). Mix by inverting 3–4 times. N o t e :Refer to the appropriate lysate preparation section in the Technical Manual #TM048 to determine whether the sample should be heated at 70°C for 3 minutes. 5.Centrifuge for 10 minutes. Transfer the cleared lysate to a fresh tube.
6.Add 2
200µl  95% e t h a n o l to cleared lysate and mix well (pipet). The Spin and Vacuum Protocols are identical up to this point.
7.Transfer mixture to Spin Basket Assembly and centrifuge for 1 minute.Discard eluate.
8.Add 6
600µl of R R N A  W a s h  S o l u t i o n  (R W A )(+ ethanol). Centrifuge for 1 minute and discard the eluate.
9.Prepare D D N a s e  i n c u b a t i o n  m i x using the table below:S o l u t i o n
V o l u m e
×
N u m b e r  o f  P r e p s
= T o t a l
Yellow Core Buffer 40µl MnCl 2, 0.09M 5µl DNase I 5µl
Mix gently (pipet); do n
n o t vortex. 10.Apply 5
tabletotal函数50µl of DNase mix to membrane. Incubate at RT for 15 minutes. 11.Add 2200µl D N a s e  S t o p  S o l u t i o n (DSA) (+ ethanol)and centrifuge for 1 minute.
12.Add 6
600µl R N A  W a s h  S o l u t i o n  (R W A );centrifuge for 1 minute. Empty.13.Add 2
250µl  R N A  W a s h  S o l u t i o n  (R W A ); centrifuge for 2 minutes. Transfer Spin Basket to Elution Tube.
14.
Add 1
100µl  N u c l e a s e -F r e e  W a t e r to membrane. Centrifuge for 1 minute to elute the RNA and store at –70°C.RT: room temperature
Centrifugation: 12,000–14,000 × g (at RT)
*Additional protocol information is available in Technical Manual #TM048,
available online at: w
w w w .p r o m e g a .c o m ORDERING /TECHNICAL INFORMATION:
www.promega • Phone 608-274-4330 or 800-356-9526 • Fax 608-277-2601
Elute RNA.
DNase Stop,  centrifuge and  wash 2X.
DNase treat.
Wash.
Transfer
lysate to Spin Basket Assembly.
Add EtOH to lysate.
2859M A 02_0A
Vacuum Protocol
Elute RNA.
Assemble Spin Basket/Collection Tube.
Wash, DNase treat.DNase Stop and  wash 2X.
Transfer lysate to Spin Basket Assembly.
Add EtOH to lysate.
2860M A 02_0A
Printed in USA. Revised 3/09.
••••••••••••••••••••••••••
N o t e :For the Vacuum Protocol, follow Steps 1–6 of the Spin Protocol.
7.Attach Vacuum Adapter with Luer-Lok ®fitting to one manifold port.Gently press SV RNA Spin Basket into adapter and transfer mixture to
Spin Basket. Apply vacuum. N
N o t e :Label Collection Tube and save for Step 13.
8.Add 9
900µl  R N A  W a s h  S o l u t i o n  (R W A ). Apply vacuum until solution has passed through. Stop vacuum source and open unused port to vent manifold. Release all vacuum pressure before continuing!9.Prepare D D N a s e  i n c u b a t i o n  m i x using the table below:S o l u t i o n V o l u m e × N u m b e r  o f  P r e p s = T o t a l
Yellow Core Buffer 40µl MnCl 2, 0.09M 5µl DNase I 5µl
Mix gently (pipet); do n
n o t vortex. 10.Apply 550µl of DNase incubation mix to membrane. Incubate at RT for 15minutes.
11.Add 2
200µl  D N a s e  S t o p  S o l u t i o n (DSA) (+ ethanol)to Spin Basket. Close open port and apply vacuum.
12.Add 9
900µl  R N A  W a s h  S o l u t i o n (RWA).Repeat wash.13.Release vacuum pressure. Place Spin Basket in Collection Tube (from Step 7). Centrifuge Spin Basket/Collection Tube for 1 minute.
14.
Transfer Spin Basket to Elution Tube, add 1
100µl  N u c l e a s e -F r e e  W a t e r and centrifuge for 1 minute. Store purified RNA at –70°C.
RT: room temperature
Centrifugation: 12,000–14,000 × g (at RT)
Additional protocol information is available in Technical Manual #TM048,
available online at: w
w w w .p r o m e g a .c o m ORDERING /TECHNICAL INFORMATION:
www.promega • Phone 608-274-4330 or 800-356-9526 • Fax 608-277-2601

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