In this experiment, cdnas prepared using each method were amplified in real time with primers specific for the 5 end of the adar gene using three different. It can be used with either sequencespecific primers, polydt15 primers, or. The synthesized cdna is ready to use in realtime pcr expression analysis of multiple genes. Rna targets from 100 bp to 12 kb can be detected with. Accuscript high fidelity 1st strand cdna synthesis kit agilent. The first strand cdna synthesis kit is used for the synthesis of the first strand cdna as the starting reaction for rtpcr. Oligodt 15 primer vial 5 for standard cdna synthesis followed by cloning or restriction enzyme. Roche diagnostics transcriptor first strand cdna synthesis kit, 100 reactions manufacturer. Hyperscript first strand synthesis kit 1 bohemia genetics.
Reverse transcriptases rts use an rna template and a short primer complementary to the 3 end of the rna to direct the synthesis of the first strand cdna, which can be used directly as a template for the polymerase chain reaction pcr. The system can be used with as little as 1 ng or as much as 5 g of total rna. The kit uses mmlv reverse transcriptase with lower rnase h activity, which avoids degradation of rna when rnadna hybrid forms in the progress of cdna synthesis. The mmlv reverse transcriptase 1ststrand cdna synthesis kit is optimized for generating fulllength firststrand cdna from total cellular rna or polya. Use 1 g of isolated mrna to generate first strand cdna for secondstrand synthesis and subsequent cloning reactions. It uses cloned moloney murine leukemia virus mmlv reverse transcriptase to synthesize first strand cdna from an mrna population. The firststrand produc t is diluted with elut ion buffer. This cdna synthesis kit provides all required reagents, except rna template and genespecific primers, to create firststrand cdna from an rna template. For robust twostep rtpcr and rt qpcr of rna up to 14 kb. Truscript first strand cdna synthesis kit for mrna product insert. Superscript iii firststrand synthesis system for rtpcr. The tonkbio first chain cdna synthesis kit for rtpcr is optimized to efficiently synthesize first chain cdna from mrna or total rna templates. June 2017 the transcriptor first strand cdna synthesis kit is designed for first strand cdna synthesis reactions up to 14 kb. First strand cdna synthesis protocols thaw kit components on ice and mix by inverting several times.
The rt 2 first strand kit provides a rapid and convenient procedure for efficient first strand cdna synthesis and genomic dna elimination in rna samples. Reverse transcriptases rts use an rna template and a primer complementary to the rna to direct the synthesis of the first strand cdna, which can be used directly as a template for the polymerase chain reaction pcr. Avoid repeated freezethaw cycles for control neo pa rna, rnase. All kit components are optimized to facilitate efficient cdna synthesis using 1 pg to 1 g of input total rna. Accuscript high fidelity 1st strand cdna synthesis kit. However, this step can be omitted in some cases unpublished results. The smarter pcr cdna synthesis kit is an improved version of. If random primer mix is used, an incubation step at 25c for 5 minutes is recommended before the 42c incubation.
The kit uses revertaid reverse transcriptase rt, a recombinant mmulv rt which maintains activity at 4250c and is suitable for synthesis of cdna up to kb. Truscript first strand cdna synthesis kit product insert. The first strand cdna synthesized with this kit can be used for variety of applications, including second strand synthesis, hybridization, pcr amplification, and real. A control reaction without reverse transcriptase is recommended to examine the dna contamination in. Cdna libraries continued second strand synthesis 19, 91, 127 size distribution42, 43, 145. Protoscript ii first strand cdna synthesis kit features two optimized mixes, protoscript ii enzyme mix and protoscript ii reaction mix. Instruction manual protoscript ii first strand cdna.
Protocol for cdna synthesis and qrtpcr cdna synthesis. Following firststrand cdna synthesis, the sample can be used directly for in vitro amplification using pcr or for second strand synthesis. Figure 2 shows that cdna prepared with the marligens first strand cdna synthesis system provides significantly enhanced sensitivity compared to cdna prepared with a competitors kit. The rnadependent dna polymerase activity is responsible for first strand cdna synthesis. After synthesis, the cdna can be amplified with specific primers by pcr without int. It uses cloned moloney murine leukemia virus mmlv reverse transcriptase to synthesize first strand cdna. Rna isolationpurification and firststrand cdna synthesis entered by kevin janes janes lab protocols 122816 2 wet pellets can be warmed for a few seconds at 37oc to increase evaporation smaller. First strand cdna synthesis kit 11801025 the use of this others has been cited in the following citations controlling obesity and metabolic diseases by hydrodynamic delivery of a fusion gene of exendin4 and. These are available as convenient pdf files online at procedure for firststrand cdna synthesis. This kit is powered by primescript reverse transcriptase primescript rt, which has exceptionally strong strand displacement activity and efficiently synthesizes cdna. Roche diagnostics transcriptor first strand cdna synthesis.
First strand cdna synthesis using the neb protoscript kit adapted from neb protocol overview reverse transcriptase is an enzyme capable of synthesizing a strand of dna the first strand complementary to the mrna template cdna. Synthesis kit is a complete system for efficient synthesis of first strand cdna from mrna or total rna templates. First strand cdna synthesis reaction denaturation of rna and primer at 6570c for 5. Firststrand cdna synthesis kit ge healthcare, 27926101. Quantification of the g6pdh gene with the transcriptor first strand cdna synthesis kit and the lightcycler 2. The resulting singlestranded cdna is suitable for use in realtime quantitative reverse transcription pcr rtqpcr. For robust twostep rtpcr and rtqpcr of rna up to 14 kb. The protoscript first strand cdna synthesis kit is contains everything needed for cdna synthesis. First strand cdna synthesis reaction denaturation of rna and primer at 70c for 5 minutes can remove secondary structures that may impede long cdna synthesis. After completion of cdna synthesis, use 12 l of the first strand reaction out of the 20 l for pcr amplification. This kit provides all components for cdna synthesis, including reverse transcriptase, rnase. Transcriptor first strand cdna synthesis kit y version. Asfs002 product summary product description the rtstar firststrand cdna synthesis kit 3 adaptor is designed to create cdna libraries from small rnas for qpcr detection. Hyperscripttm first strand synthesis kit is ideally organized for synthesizing reaction of first strand cdna from purified mrna or total rna.
This kit provides all components for cdna synthesis, including reverse transcriptase, rnase inhibitor, oligo dt, random hexamer, dntps and nuclease free water. The first strand cdna product generated is up to 10 kb. Once the sequence of the forward primer has been determined, the primer should be ordered through a custom oligo supplier. Transcriptor first strand cdna synthesis kit roche life science. Thermo scientific revertaid first strand cdna synthesis kit is a complete system for efficient synthesis of first strand cdna from rna templates. Either the transcriptor first strand cdna synthesis kit blue curves or another supplierss reverse transcriptase red curves was used to perform reverse transcription on 100 ng to 10 pg of total rna from k562 cells. Firststrand cdna synthesis for quantitative rtpcr origene. Oligodt 15 primer vial 5 for standard cdna synthesis.
This kit is suitable for the synthesis of first strand cdna. Recombinant mmulv reverse transcriptase generates cdna up to 10 kb supplied with oligodt. Different amounts of total rna from k562 cells were reverse transcribed using random hexamer primers and the reaction conditions outlined in the pack insert of the kit. Nzy first strand cdna synthesis kit is a system that includes all the necessary components to synthesize first strand cdna, except the template rna. Figure 1 is a summary of the two procedures that may be. Thermo scientific revertaid h minus first strand cdna synthesis kit is a complete system for efficient synthesis of first strand cdna from mrna or total rna reverse transcriptase, which has a point mutation that completely eliminates rnase h activity.
Enhanced avian first strand synthesis kit components for. Mmlv reverse transcriptase 1ststrand cdna synthesis kit lucigen. The kit includes superscript iv reverse transcriptase rt, a proprietary mmlv mutant that produces robust and reliable cdna synthesis. Thermo scientific reverse transcription thermo fisher. The length of cdna achieved is verified by detection of a 9. Preparing cdna from rna samples firststrand synthesis first strand cdna synthesis employs the clontech smarter pcr cdna synthesis kit. The transcriptor first strand cdna synthesis kit is designed to reverse transcribe rna mrna, total rna, viral rna, and in vitrotranscribed rna from a variety of sources for the following applications. A third activity is nuclease activity including rnase h, which degrades rna when it is present in an rnadna hybrid molecule. Using the fermentas first strand cdna synthesis kit quantify we need 0. Store the remaining first strand cdna sample at 20 c until use. Truscript first strand cdna synthesis kit for mrna protocol. Firststrand cdna synthesis kit for realtime pcr product code pack size 75780 50 reactions 20 l usb firststrand cdna synthesis kit for realtime pcr fig.
It can be used with either sequencespecific primers, polydt15 primers, or random primers, pdn6. First strand cdna synthesis protocols e6300 introduction. If the cdna is used for downstream qpcr, the reverse transcription reaction can be completed in 15 minutes. Reverse transcriptases rts use an rna template and a primer complementary to the rna to direct the synthesis of the first strand cdna. The transcriptor first strand cdna synthesis kit is designed to reverse transcribe rna mrna, total rna, viral rna, and in vitro transcribed rna from a. The performance of abscript ii first strand cdna synthesis kit is tested in an rt reaction using jurkat total rna with primer dt 23 vn. Amv reverse transcritase is used for first strand cdna synthesis. First strand cdna synthesis after thawing, mix and briefly centrifuge the components of the kit.
The efficiency and yield of cdna first strand synthesis are higher, and the first strand of cdna can be synthesized using pg total rna or mrna. Therefore, degradation of rna does not occur during first strand. The ncode mirna firststrand cdna synthesis kits and qrtpcr kits provide qualified reagents for the polyadenylation of micrornas mirnas from total rna and synthesis of first strand cdna from the tailed mirnas for use in realtime quantitative pcr qpcr. First strand cdna synthesis kit enhanced avian str1 is a reverse transcriptasepolymerase chain reaction rtpcr kit that utilizes a highly purified avian myeloblastosis virus reverse transcriptase. Reverse transcriptase also has some dnadependent dna polymerase activity. The synthesis of dna from an rna template, via reverse transcription, produces complementary dna cdna. Lightcycler faststart dna masterplus sybr green i, was then used with primers for g6pdh to quantify geneexpression levels. Firststrand cdna synthesis kit is designed to generate fulllength first strand cdna from mrna templates. Assay sensitivity and dynamic range various amounts of hela cell total rna, ranging from 850 ng down to 10 pg, were reverse transcribed using anchored oligodt 23 vn as. Rna isolationpurification and firststrand cdna synthesis.
Mix the following components and incubate at 42c for 1 hour. This kit provides all components for cdna synthesis. We recommend incubation at 42c for one hour for maximum yield and length. This kit is powered by primescript reverse transcriptase primescript rt, which has exceptionally strong strand displacement activity.
Primescript 1st strand cdna synthesis kit user manual takara bio. Nzy firststrand cdna synthesis kit cdna kits nzytech. Usb firststrand cdna synthesis kit for realtime pcr. This is recommended when the cdna synthesis reaction uses less than 10 ng of rna input.
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