Quick Protocol: Ovation RNA Amplification System V2

Quick Protocol: Ovation RNA Amplification System V2
Quick Protocol
Ovation RNA Amplification System V2
®
Enter the following information to automatically calculate the volumes needed to prepare each reaction.
The calculated volumes include an appropriate overfill in excess of the nominal volume requirements
to allow for volume loss due to handling. Simply print this document to create a working guide for your
experiment, which can be kept as a record.
Operator’s Name:
Date:
Ovation Kit Part No.
Ovation Kit Lot No.
Number of Samples:*
Thermal Cycler Programs
First Strand cDNA Synthesis
Program 1: Primer Annealing
65°C – 5 min, hold at 4ºC
Program 2: First Strand Synthesis
48°C – 60 min, 70°C – 15 min, hold at 4ºC
SECOND Strand cDNA Synthesis
Program 3: Second Strand Synthesis
37°C – 30 min, 75°C – 15 min, hold at 4ºC
SPIA Amplification & Modification
Program 4: SPIA® Amplification
48°C – 60 min, 95°C – 5 min, hold at 4ºC
* Number of samples field ties into embedded logic to calculate master mix volumes, number of reactions.
Q u i c k P r otoco l
First Strand cDNA Synthesis
Obtain Nuclease-Free Water D1 (green) from -20ºC and leave at room temp.
Thaw the First Strand Reagents (blue). Mix each reagent, spin and place on ice.
For each assay place 5 μL of total RNA into 0.2 mL PCR tube and place on ice.
Add 2 μL of First Strand Primer Mix A1, flick tubes to mix and spin.
Place the tubes in a thermal cycler running Program 1 (65°C – 5 min, hold at 4ºC).
After 5 minutes at 65°C, immediately snap cool tubes on ice.
Prepare First strand master mix (calculation allows for appropriate overfill). Please
be sure to pipet a3 enzyme slowly and rinse out tip at least five times into buffer.
Per sample combine:
12 μL Buffer Mix a2 ver 4 + 1 μL Enzyme Mix a3 ver 7.
mix well.
No. of Samples
A2
A3
1
12 µL
1 µL
0
0
0
No. of Samples
B1
B2
1
18 µL
2 µL
0
0
0
Add 13 µL of the First Strand Master Mix to each tube, mix and spin.
Place the tubes in a thermal cycler running Program 2
(48°C – 60 min, 70°C – 15 min, hold at 4ºC).
Second Strand cDNA Synthesis
Thaw the Second Strand Reagents (yellow). Mix each reagent,
spin and place on ice.
Once the thermal cycler reaches 4°C, remove tubes, spin and place on ice.
Make Second Strand Master Mix (calculation allows for appropriate overfill).
Per sample combine:
18 µL Buffer Mix B1 ver 4 + 2 µL Enzyme Mix B2 ver 1.
Mix well.
Add 20 µL of Second Strand Master Mix to each first strand reaction tube,
mix and spin.
Place the tubes in a thermal cycler running Program 3
(37°C – 30 min, 75°C – 15 min, hold at 4ºC).
Once the thermal cycler reaches 4ºC, spin and place tubes on ice.
Q u i c k P r otoco l
SPIA® Amplification
Thaw the SPIA Amplification Reagents (red). Vortex C1
and C2, invert C3 5 times. Spin all, place on ice.
Make SPIA Master Mix (calculation allows for appropriate
overfill). Please be sure to pipet C3 enzyme slowly and rinse
out tip at least five times into buffer. Per sample combine:
72 μL C2 ver 6+ 4 μL C1 ver 1 + 4 μL D1 (water)
+ 40 μL C3 ver 5.
Mix well.
No. of Samples
C2
C1
Water D1
C3
1
72 µL
4 µL
4 µL
40 µL
0
0
0
0
0
On ice, add 120 µL of SPIA Master Mix to each second
strand reaction tube, mix and spin.
Place half of the 160 μL reaction into a separate 0.2 mL PCR
tube, cap tightly and spin.
Place tubes in a thermal cycler running Program 4
(48°C – 60 min, 95°C – 5 min, hold at 4ºC).
Once the thermal cycler reaches 4ºC, spin and place tubes
on ice.
Proceed immediately to purification step or store SPIA
cDNA at -20ºC.
Purification of Amplified SPIA cDNA
Purification Kit Part No.
Refer to the user guide and follow your method of
choice for purification:
Purification Kit Lot No.
Add Binding Buffer in volume of:
Spin at speed:
For a duration of:
Add Wash Buffer in volume of:
Spin at speed:
For a duration of:
Repeat for second wash.
To elute sample use Nuclease-Free Water D1 provided with the Ovation Kit.
Add Nuclease-Free Water D1 in volume of:
Spin at speed:
For a duration of:
©2016 NuGEN Technologies, Inc. All rights reserved. The Encore®, Ovation® and Applause® families of Products and methods of
their use are covered by several issued U.S. and International patents and pending applications (www.nugen.com). NuGEN, Ovation,
SPIA, Ribo-SPIA, Applause, Encore, Prelude, Mondrian and Imagine More from Less are trademarks or registered trademarks of
NuGEN Technologies, Inc. Other marks appearing in these materials are marks of their respective owners.
For Research Use Only.
M01142 v3
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