Random primers for cdna synthesis - Order Custom Essay
Affymetrix hybridizations require 10–15 ug of labeled probe. Because RNA in this amount is rarely available from FACS isolated cells, we amplify initial RNA preparations with two rounds of a T7 RNA polymerase-dependent amplification protocol. The following bench protocol produces high-quality cRNA from 100 ng of total RNA. This protocol is a modified version of an amplification strategy originally described by Affymetrix (GeneChip Eukaryotic Small Sample Target Labeling Technical Note, Part 701129 Rev 1).
PrimeScript Reverse Transcriptase synthesizes long 1st-strand cDNA ..
Add 1 μl of 1 μg/μl T7-promoter linked oligo dT to 12 μl of the single stranded cDNA and heat for 3 min at 95–100 °C to degrade RNA. Chill on ice and then add
Add 2 μl T4 DNA polymerase (NEB, 3 unit/μl) and continue the incubation at 16°C for 30 minutes. Add 80 μl phenol/chloroform (25:24) mix, vortex, and spin at 14,000 rpm in an Eppendorf centrifuge at room temperature for 3 minutes. Transfer the supernatant to a new tube. Repeat extraction once. Recover the double-stranded cDNA from reaction solution, using 10 ng DstNI digested pBR322 as carrier, by GENECLEAN II (BIO 101) according to manufacturer's instruction. Elute cDNA into 29 μl Tris (pH 8.0).
of RNA into all two-step cDNA synthesis ..
PrimeScript Reverse Transcriptase has exceptionally strong strand displacement activity and enables efficient preparation of cDNA up to 12 kb in length. It is robust, versatile and well-suited for applications requiring full-length cDNA such as preparation of cDNA libraries and other techniques involving first strand cDNA synthesis (RT-PCR, preparation of cDNA probes, real-time quantitative RT-PCR). PrimeScript RTase can be used for performing a reverse transcription reaction with any RNA template including GC-rich templates and RNAs with high levels of secondary structure. This enzyme is a modified, recombinant MMLV (Moloney Murine Leukemia Virus) reverse transcriptase and is verified to be RNase H Minus. Because of the excellent extension capability of PrimeScript Reverse Transcriptase, preparation of cDNA can be performed at a lower temperature (42°C), decreasing the risk of RNA degradation that can occur during conventional reactions performed at higher temperatures.
Compare cDNA Synthesis Kits from leading ..
Both the pd(N)6 (80 nmol) and pd(N)9 (50 nmol) random primers contain approximately 150 µg (5 OD) and are ideal for use in cDNA synthesis, RT-PCR and hybridization applications.
cDNA Synthesis; cDNA Synthesis Kits; ..
qScript cDNA SuperMix is stable for up to 1 year when stored in a constant temperature freezer at -20°C. The kit may be stored at -80°C to further extend the product’s shelf life. SuperMix reagent shows no loss in functional performance after 20 cycles of freezing on dry ice and thawing on ice.
First Strand cDNA Synthesis Kit for RT-PCR (AMV) | …
First-strand cDNAs were synthesized following each manufacturers protocol with 1 µ or 1 ng of HeLa cell total RNA as template. 1/10th of first-strand reactions were used for qRT-PCR of 5'-end region of TRRAP gene with PerfeCTa™ SYBR Green SuperMix. Averaged plots from 4 replicate qPCR reactions are shown.