Bio-Rad iScript Select cDNA Synthesis Kit User Manual
iScript™Select cDNA Synthesis Kit
25 x 20 µl reactions 170-8896
100 x 20 µl reactions 170-8897
For research purposes only
Store at -20°C
The iScript Select cDNA synthesis kit is a sensitive, flexible, and easy-to-use kit for the
generation of first-strand cDNA. This robust reverse transcription kit allows a selection of
first-strand priming strategies
†
, oligo(dT) primers only, random primers only, or user-designed
gene-specific primers.
This cDNA synthesis kit provides all required reagents, except RNA template and
gene-specific primers, to create first-strand cDNA from an RNA template. All kit components
are optimized to facilitate efficient cDNA synthesis using 1 pg to 1 µg of input total RNA.
The iScript reverse transcriptase mixture contains a recombinant RNase H+ MMLV reverse
transcriptase preblended with a recombinant RNase inhibitor. The activity of this reverse
transcriptase is optimized for use with the 5x iScript select reaction mix. This unique blend
of buffers, stabilizers, and dNTPs streamlines reaction setup and ensures robust synthesis
of first-strand cDNA.
To enhance first-strand priming, the iScript select cDNA sythesis kit incorporates a
proprietary enhancer solution into the primer-template hybridization step. To simplify
reaction setup, this enhancer is preblended with the oligo(dT) primers and random primers
provided in the kit. Consequently, there is no need to add enhancer solution to reactions
using the provided primer mixes. However, when using a gene-specific primer (GSP), the
enhancer solution must be added to the reaction. A separate protocol and tube labeled
GSP Enhancer Solution is included for this purpose. The addition of enhancer solution to
cDNA reactions can significantly improve yields, resulting in earlier detection in real-time
PCR.
Storage and Stability
Store kit components at –20°C in a constant-temperature freezer. When stored under
these conditions, kit components are stable for a minimum of one year after the shipping
date. Nuclease-free water can be stored at room temperature.
†
To perform first-strand synthesis with a mixture of random primers and oligo(dT) primers, the iScript cDNA
synthesis kit (170-8890 or 170-8891) with an optimized, preblended mixture of random primers and oligo(dT)
primers is recommended.
Recommendations for Optimal Results Using the iScript Select cDNA
Synthesis Kit
The maximum amount of the cDNA reaction that is recommended for downstream PCR is
one-tenth of the reaction volume, typically 2
µl.
When using larger amounts of input RNA (>1 µg), the reaction should be scaled up; e.g.,
40 µl reaction for 2 µg, or 100 µl reaction for 5 µg to ensure optimum synthesis efficiency.
Related Amplification Products From Bio-Rad Laboratories
Reagents for PCR or Real-Time PCR
iTaq™ DNA Polymerase 170-8870
iQ™ Supermix 170-8860
iQ SYBR®Green Supermix 170-8880
iTaq Supermix With ROX 170-8854
iTaq SYBR Green Supermix With ROX 170-8850
iScript cDNA Synthesis Kit 170-8890
iScript One Step RT-PCR Kit With SYBR Green 170-8892
iScript One Step RT-PCR Kit for Probes 170-8894
iProof™ High-Fidelity DNA Polymerase 172-5301
Other Reagents
dNTP Mix, 200 µl, 10 mM each dNTP 170-8874
(dATP, dCTP, dGTP, dTTP)
MgCl2Solution, 50 mM, 1.25 ml 170-8872
Aerosol Barrier Pipet Tips
Xcluda™ Style B Tips 211-2006
Nuclease-Free Tubes
0.2 ml Thin-Wall Tubes TWI-0201
0.2 ml Thin-Wall Plates HSP-9601 (Low-Profile)
HSS-9601 (Full Height)
RNA Purification Kits
Aurum™ Total RNA Mini Kit 732-6820
Aurum Total RNA Kit, 2 x 96-well 732-6800
Aurum Total RNA Fatty and Fibrous Tissue Kit 732-6830
PureZOL™ RNA Isolation Reagent 732-6890
For ordering information on larger pack sizes, or to learn more about Bio-Rad amplification
reagents and instruments, visit www.bio-rad.com/amplification/
Bio-Rad Laboratories, Inc. is licensed by Molecular Probes, Inc. to sell reagents containing SYBR Green I for use in
real-time PCR, for research purposes only. SYBR Green is a registered trademark of Molecular Probes, Inc.
iTaq, iQ, iScript, iProof, Xcluda, Aurum, and PureZOL are trademarks of Bio-Rad Laboratories.
10001023 Rev B
Bio-Rad Laboratories, Inc.
2000 Alfred Nobel Drive, Hercules, CA 94547
510-741-1000
Kit Contents
25 Reactions 100 Reactions
Reagent Volume Volume Description
iScript reverse 25 µl 100 µl RNase H
+
MMLV reverse transcriptase
transcriptase and RNase inhibitor protein
5x iScript select 400 µl 400 µl 5x reaction buffer containing dNTPs,
reaction mix magnesium chloride, and stabilizers
Oligo(dT)
20
200 µl 200 µl Purified oligo(dT)20primer in a
primer mix proprietary enhancer solution
Random primer 200 µl 200 µl Purified random primers in a proprietary
mix enhancer solution
GSP enhancer 200 µl 200 µl Proprietary solution for reactions using
solution gene-specific primers
Nuclease-free 1.5 ml 1.5 ml
water
Reaction Setup With Oligo(dT) Primers or Random Primers
Important Note – Please Read Before Starting
This protocol is for use with either oligo(dT) or random primers. Only use the provided primers.
Use of primers from other sources can adversely affect performance and sensitivity. To use
gene-specific primers, please follow the protocol Reaction Setup With Gene-Specific Primers.
Protocol for Oligo(dT) or Random Primers
1. Thaw all components except iScript reverse transcriptase. Mix thoroughly and briefly
centrifuge to collect contents to the bottom of the tube before using. Place
components on ice.
2. Add the following components to a 0.2 ml PCR tube or each well of a 96-well PCR
reaction plate on ice:
Components
Volume
Nuclease-free water Variable
5x iScript select reaction mix 4 µl
Oligo(dT)
20
primer or random primer 2 µl
RNA sample (1 pg to 1 µg total RNA) Variable
iScript r
everse transcriptase 1 µl
Total 20 µl
Note: for multiple reactions, prepare a master mix with the above components, except
RNA, and then dispense to each reaction.
3. Mix gently and incubate as follows:
For oligo(dT)-primed cDNA reactions, incubate for 60–90 min at 42°C.
For random-primed cDNA reactions, incubate for 5 min at 25°C, then 30 min at
42°C.
4. Incubate at 85°C for 5 min to heat-inactivate the reverse transcriptase.
5. Store cDNA product at –20°C to +4°C.
6. The resulting cDNA product can be used directly for PCR amplification. Typically,
one-tenth (2 µl) of the first-strand reaction provides sufficient target for most PCR
applications. Optionally, the cDNA can be diluted in TE buffer [10 mM Tris (pH 8.0),
0.1 mM EDTA] for addition of larger volumes (5–10 µl) to PCR reactions.
Reaction Setup With Gene-Specific Primers
Important Note – Please Read Before Starting
This protocol is for use with user-defined gene-specific primers. For random or oligo(dT) primers,
please follow the protocol Reaction Setup With Oligo(dT) Primers or Random Primers.
Protocol for Gene-Specific Primers
1. Thaw all components except iScript reverse transcriptase. Mix thoroughly and briefly
centrifuge to collect contents to the bottom of the tube before using. Place
components on ice.
2. Add the following components to a 0.2 ml PCR tube or each well of a 96-well PCR
reaction plate on ice:
Components
V
olume
Nuclease-free water Variable
5x iScript select reaction mix 4 µl
Gene-specific primer (2–10 pmol) Variable (100–500 nM in
20 µl final volume)
GSP enhancer solution 2 µl
RNA sample (1 pg to 1 µg total RNA) Variable
iScript r
everse transcriptase 1 µl
Total 20 µl
Note: for multiple reactions, prepare a master mix with the above components, except
RNA, and then dispense to each reaction.
3. Mix gently and incubate at 42°C for 30–60 min.
As required, incubation times can be extended to create longer cDNAs for cloning
purposes.
4. Incubate at 85°C for 5 min to heat-inactivate the reverse transcriptase.
5. Store cDNA product at –20°C to +4°C.
6. The resulting cDNA product can be used directly for PCR amplification. Typically,
one-tenth (2 µl) of the first-strand reaction provides sufficient target for most PCR
applications. Optionally, the cDNA can be diluted in TE buffer [10 mM Tris (pH 8.0),
0.1 mM EDTA] for addition of larger volumes (5–10 µl) to PCR reactions.
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