Select cDNA Synthesis Kit iScript - Bio-Rad

1 iScript Select cDNA Synthesis Kit25 x 20 l reactions170-8896100 x 20 l reactions170-8897 For research purposes onlyStore at -20 CThe iScript Select cDNA Synthesis kit is a sensitive, flexible, and easy-to-use kit for thegeneration of first-strand cDNA. This robust reverse transcription kit allows a selection offirst-strand priming strategies , oligo(dT) primers only, random primers only, or user-designedgene-specific 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 componentsare optimized to facilitate efficient cDNA Synthesis using 1 pg to 1 g of input total iScript reverse transcriptase mixture contains a recombinant RNase H+ MMLV reversetranscriptase preblended with a recombinant RNase inhibitor. The activity of this reversetranscriptase is optimized for use with the 5x iScript Select reaction mix.

2 This unique blendof buffers, stabilizers, and dNTPs streamlines reaction setup and ensures robust synthesisof first-strand enhance first-strand priming, the iScript Select cDNA sythesis kit incorporates aproprietary enhancer solution into the primer-template hybridization step. To simplifyreaction setup, this enhancer is preblended with the oligo(dT) primers and random primersprovided in the kit. Consequently, there is no need to add enhancer solution to reactionsusing the provided primer mixes. However, when using a gene-specific primer (GSP), theenhancer solution must be added to the reaction. A separate protocol and tube labeledGSP Enhancer Solutionis included for this purpose. The addition of enhancer solution tocDNA reactions can significantly improve yields, resulting in earlier detection in real-timePCR. Storage and StabilityStore kit components at 20 C in a constant-temperature freezer.

3 When stored underthese conditions, kit components are stable for a minimum of one year after the shippingdate. 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 cDNAsynthesis 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 cDNAS ynthesis KitThe maximum amount of the cDNA reaction that is recommended for downstream PCR isone-tenth of the reaction volume, typically 2 using larger amounts of input RNA (>1 g), the reaction should be scaled up; ,40 l reaction for 2 g, or 100 l reaction for 5 g to ensure optimum Synthesis Amplification Products From Bio-Rad LaboratoriesReagents for PCR or Real-Time PCRiTaq DNA Polymerase170-8870 iQ Supermix170-8860 iQ SYBR Green Supermix170-8880iTaq Supermix With ROX170-8854iTaq SYBR Green Supermix With ROX170-8850iScript cDNA Synthesis Kit170-8890iScript One Step RT-PCR Kit With SYBR Green170-8892iScript One Step RT-PCR Kit for Probes170-8894iProof High-Fidelity DNA Polymerase172-5301 Other ReagentsdNTP Mix, 200 l, 10 mM each dNTP170-8874(dATP, dCTP, dGTP, dTTP)MgCl2 Solution, 50 mM, ml170-8872 Aerosol Barrier Pipet TipsXcluda Style B Tips211-2006 Nuclease-Free ml Thin-Wall ml Thin-Wall PlatesHSP-9601 (Low-Profile)HSS-9601 (Full Height)

4 RNA Purification KitsAurum Total RNA Mini Kit732-6820 Aurum Total RNA Kit, 2 x 96-well732-6800 Aurum Total RNA Fatty and Fibrous Tissue Kit732-6830 PureZOL RNA Isolation Reagent732-6890 For ordering information on larger pack sizes, or to learn more about Bio-Rad amplificationreagents and instruments, visit Laboratories, Inc. is licensed by Molecular Probes, Inc. to sell reagents containing SYBR Green I for use inreal-time PCR, for research purposes only. SYBR Green is a registered trademark of Molecular Probes, , iQ, iScript , iProof, Xcluda, Aurum, and PureZOL are trademarks of Bio-Rad Rev BBio-Rad Laboratories, Alfred Nobel Drive, Hercules, CA 94547510-741-1000 Kit Contents25 Reactions 100 ReactionsReagentVolumeVolumeDescriptioni Script reverse25 l100 lRNase H+MMLV reverse transcriptasetranscriptaseand RNase inhibitor protein5x iScript select400 l400 l5x reaction buffer containing dNTPs,reaction mixmagnesium chloride, and stabilizersOligo(dT)20200 l200 lPurified oligo(dT)20primer in a primer mixproprietary enhancer solutionRandom primer200 l200 lPurified random primers in a proprietarymixenhancer solutionGSP enhancer200 l200 lProprietary solution for reactions usingsolutiongene-specific mlwaterReaction Setup With Oligo(dT)

5 Primers or Random PrimersImportant Note Please Read Before StartingThis protocol is for use with either oligo(dT) or random primers. Only use the provided 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 for Oligo(dT) or Random Primers1. Thaw all components except iScript reverse transcriptase. Mix thoroughly and brieflycentrifuge to collect contents to the bottom of the tube before using. Placecomponents on Add the following components to a ml PCR tube or each well of a 96-well PCRreaction plate on ice:ComponentsVolumeNuclease-free waterVariable5x iScript Select reaction mix4 lOligo(dT)20primer or random primer2 lRNA sample (1 pg to 1 g total RNA)VariableiScript reverse transcriptase1 lTotal20 lNote: for multiple reactions, prepare a master mix with the above components, exceptRNA, and then dispense to each Mix gently and incubate as follows:For oligo(dT)-primed cDNA reactions, incubate for 60 90 min at 42 random-primed cDNA reactions, incubate for 5 min at 25 C, then 30 min at42 Incubate at 85 C for 5 min to heat-inactivate the reverse Store cDNA product at 20 C to +4 The resulting cDNA product can be used directly for PCR amplification.

6 Typically, one-tenth (2 l) of the first-strand reaction provides sufficient target for most PCRapplications. Optionally, the cDNA can be diluted in TE buffer [10 mM Tris (pH ), mM EDTA] for addition of larger volumes (5 10 l) to PCR Setup With Gene-Specific PrimersImportant Note Please Read Before StartingThis 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 for Gene-Specific Primers1. Thaw all components except iScript reverse transcriptase. Mix thoroughly and brieflycentrifuge to collect contents to the bottom of the tube before using. Placecomponents on Add the following components to a ml PCR tube or each well of a 96-well PCRreaction plate on ice:ComponentsVolumeNuclease-free waterVariable5x iScript Select reaction mix4 lGene-specific primer (2 10 pmol)Variable(100 500 nM in 20 l final volume)GSP enhancer solution2 lRNA sample (1 pg to 1 g total RNA)VariableiScript reverse transcriptase1 lTotal20 lNote: for multiple reactions, prepare a master mix with the above components, exceptRNA, and then dispense to each Mix gently and incubate at 42 C for 30 60 required, incubation times can be extended to create longer cDNAs for Incubate at 85 C for 5 min to heat-inactivate the reverse Store cDNA product at 20 C to +4 The resulting cDNA product can be used directly for PCR amplification.

7 Typically, one-tenth (2 l) of the first-strand reaction provides sufficient target for most PCRapplications. Optionally, the cDNA can be diluted in TE buffer [10 mM Tris (pH ), mM EDTA] for addition of larger volumes (5 10 l) to PCR reactions.