Science Catalog 2012 Life Cell Signaling ImProm-II™ Reverse Transcription System Product ImProm-II™ Reverse Transcriptase For Laboratory Use. Description: The ImProm-II™ Reverse Transcription System produces efficient, robust synthesis of first-strand cDNA in preparation for PCR amplification. The components of the ImProm-II™ Reverse Transcription System can be used to reverse transcribe RNA templates starting with total RNA, poly(A)+ mRNA or synthetic transcript RNA. The optimized reaction buffer and powerful ImProm-II™ Reverse Transcriptase provided in the ImProm-II™ System together enable robust, full-length cDNA synthesis for the reproducible analysis of rare or long messages. The cDNA synthesis conditions have been formulated for standalone applications or for easy transition to gene-specific target amplification. From 1–20μl of the reverse transcription reaction can be directly amplified using Taq DNA polymerase in coupled or uncoupled PCR. Features: • Full-Length RT-PCR: Reverse transcribe long RNA template up to 8.9kb. • Microarray-Compatible: May be used for incorporation of regular, Cy®3modified, Cy®5-modified, and amino-allyl-modified nucleotides. • Easy to Use: System provides all reagents for efficient reverse transcription. • Scalable and Flexible: Use 1–20μl from the initial RT reaction in subsequent PCR; the optimized buffer allows for coupled RT-PCR. • RT Provided with 5X Reaction Buffer: 250mM Tris-HCl (pH 8.3 at 25°C), 375mM KCl, 50mM DTT. A 25mM MgCl2 Solution also is included. • Versatile: Use with your thermostable DNA polymerase of choice. Storage Conditions: Store at –20°C. Store Positive Control RNA at –70°C. Protocol ImProm-II™ Reverse Transcription System Technical Manual First-strand cDNA completed by ImProm-II™ RT TTTTTTTT 37°C M – 940bp 42°C45°C50°C 8.9kb APC gene mRNA 55°C Part# TM236 0 ImProm-II™ System SuperScript® System -II 0 ImProm-II™ System SuperScript® System -II Relative Cy®3 and Cy®5 nucleotide incorporation by ImProm-II™ Reverse Transcription System in comparison to Superscript® II First Strand Synthesis System. Results with Cy®3 dUTP (Panel A) and Cy®5 dUTP (Panel B) incorporation are reported. Panels A and B correspond to Panels A and B in the figure above. 50 10 Size Cat.# Price (Fr) ImProm-II™ Reverse Transcription System 100 reactions A3800 729.00 Available Separately 10 reactions A3801 43.00 100 reactions A3802 301.00 500 reactions A3803 1280.00 A. Cy®3 Incorporation ImProm-II™ RT SuperScript® II RT B. Cy®5 Incorporation ImProm-II™ RT SuperScript® II RT Incorporation studies of fluorescently labeled nucleotides. ImProm-II™ Reverse Transcription System allows for high-efficiency incorporation of Cy®3 and Cy®5 fluorescent nucleotides. This demonstrates fluorescent nucleotide incorporation by ImProm-II™ RT vs. SuperScript® II RT using a 1.2kb kanamycin transcript as template. A single fluorescent band is produced and visualized using an FMBIO® II Fluorescence Imaging System. A. Cy®3 Incorporation 100 B. Cy®5 Incorporation 20 Full-length cDNA synthesis of 8.9kb template over a range of temperatures using the ImProm-II™ Reverse Transcription System as demonstrated by selective amplification of terminal 3´ sequences in two-step RT-PCR. Entire 8.9kb message must be reverse transcribed by the ImProm-II™ RT from the oligo(dT) primer to amplify the terminal 940bp sequence. Message was amplified from either 1μg or 100ng of total RNA. Control reactions without the reverse transcriptase are shown (–RT) as well. Details of experiment available in the ImProm-II™ Reverse Transcription System Technical Manual, #TM236. A. B. A comparison of typical 21-by-21 feature array blocks hybridized with ImProm-II™- or SuperScript®-II-generated fluorescent cDNA probes. Panel A. Array hybridized with ImProm-II™-generated fluorescent cDNA probes. Panel B. Array hybridized with SuperScript®-II-generated fluorescent cDNA probes. Image brightness was normalized to yield similar background intensities. See Kasler et al. Promega Notes 81, 14–15, for experimental details. 216 For complete and up-to-date product information visit: www.promega.com/catalog 1∝g 100ng –RT 1∝g 100ng –RT 1∝g 100ng –RT 1∝g 100ng –RT 1∝g 100ng –RT 3291TB07_2A Relative Fluorescence Units (in thousands) 3698TA04_2A 3395MB04_2A 3392TC05_1A Pagina 219
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