Bio-Rad iScript™ Select cDNA Synthesis Kit - Manual (Page 2)

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)
20
primer 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 reverse 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 Volume
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 reverse 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.