![]() SuperScript® III Reverse Transcriptase is a version of M-MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability. Amplicon size-compatible detection of targets up to 4.5 kb in length for greater flexibility.Specificity-uses SuperScript® III RT for cDNA synthesis up to 55☌, for more specific priming with gene specific primers (see figure).Convenience-one-step format for speed, convenience, and less reaction-to-reaction variability.Sensitive-routine detection down to 0.01 pg total RNA (see figure).The amount of starting material can range from 0.01 pg to 1 µg of total RNA. The system uses a mixture of SuperScript® III Reverse Transcriptase and Platinum® Taq DNA polymerase in an optimized reaction buffer, and it can detect a wide range of RNA targets, from 200 bp to 4.5 kb. Using this convenient one-step formulation, you can perform both cDNA synthesis and PCR amplification in a single tube using gene-specific primers, and target RNAs from either total RNA or mRNA. I highly recommend this product.The SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase is designed for the sensitive, reproducible, end-point detection and analysis of RNA molecules by RT-PCR. I get very good yields of cDNA and the easy-to-follow instruction manual makes this kit a great resource in our lab. The Invitrogen SuperScript™ II RT System allows me to make cDNA from my isolated total RNA quickly and efficiently. We are interested in the function of Heat Shock Factor (HSF) as an activator in wild type and long-lived C. ![]() I then use the cDNA in quantitative RT-PCR (qRT-PCR) assays (using the Chromo 4 from Bio-Rad Laboratories). The whole procedure is very quick, taking only a couple of hours. RNase H is then added and the reaction is incubated for 20 min at 37 ✬ before measuring the DNA concentration. The reaction is terminated after 15 min by placing the reaction at 70 ✬. Next, the SuperScript™ II RT is added and the reaction is incubated at 42 ✬ for 50 min. Then, I add RT Buffer, MgCl 2, DTT and RNaseOUT, and incubate at 42 ✬ for 2 min. Basically, I mix my RNA with dNTP, Oligo(dT), and DEPC-treated water and incubate at 65 ✬ for 5 min. The manual that comes with the kit is very easy to follow. A control RNA is included in the kit to verify performance, but I have not been using it. This increases sensitivity of PCR from cDNA. The kit also provides RNase H, which removes the RNA template from the cDNA:RNA hybrid molecules after the first-strand synthesis. This method for priming first-strand synthesis is recommended when performing RT-PCR from a new mRNA target gene because oligo(dT) produces an RT-PCR product more consistently than other methods, such as random hexamers or gene-specific primers. I prime my first-strand cDNA synthesis reaction using the oligo(dT) provided in the kit, which hybridizes to 3’ poly(A) tails. It also has increased thermal stability and so is effective up to 50 ✬. SuperScript™ II RT is not inhibited by ribosomal RNA and transfer RNA, so it can effectively synthesize first-strand cDNA from a total RNA preparation. The first-strand cDNA synthesis is catalyzed by SuperScript™ II RT, which has been engineered to eliminate RNase H activity, yielding greater full-length first-strand cDNA. I then use the cDNA to perform TaqMan® Real-Time PCR experiments. I have been using 2.5 ug of total RNA per reaction and I get about 20-40 ug of cDNA, as measured by spectrophotomety. The components of this kit include oligo(dT), 10X RT buffer, MgCl 2, DTT, dNTP Mix, SuperScript™ II RNase H- Reverse Transcriptase (RT), RNaseOUT recombinant ribonuclease inhibitor, RNase H and DEPC-treated water. Each reaction can convert 1 ng – 5 ug of total RNA into first-strand cDNA. The SuperScript™ Kit comes with the necessary components to perform about 50 reactions. elegans mutant strains the RNA was prepared using TRIzol Reagent. Currently, I am utilizing the Invitrogen SuperScript™ First-Strand Synthesis System for cDNA synthesis from RNA isolated from different C. ![]()
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