I. DESCRIPTIONSUPERSCRIPT™ II RNase H Reverse Transcriptase (U.S. Patent 5,244,797)is purified to near homogeneity from E. coli containing the pol gene ofMoloney Murine Leukemia Virus (1,2). The enzyme is used to synthesizefirst strand cDNA and will generally give higher yields of cDNA and morefull length product than other reverse transcriptases.USES APPLICATIONS:1. The enzyme can be used to synthesize first strand cDNA and will generally result in higher yields of cDNA and more full length product.2. Second strand synthesis if template is SS DNA. (Not recommended for 2nd strand synthesis for cDNA library construction, even after extensive RNase H treatment.3. Dideoxy sequencing of RNA or DNA.4. Primer extension.5. Synthesis of high specific activity cDNA.II. COMPONENTS18064-014 SUPERSCRIPT™ IIY00146 5X First Strand BufferY00147 0.1 M DTTIII. PROTOCOLSFirst Strand cDNA Synthesis Using SUPERSCRIPT™ II for RT-PCR:A 20-ul reaction volume can be used for 1-5 ug of total RNA or 50-500ng of mRNA. Add the following components to a nuclease-freemicrocentrifuge tube:1 ul Oligo (dT)12-18 (500 ug/ml)*1-5 ug total RNASterile, distilled water to 12 ulHeat mixture to 70°C for 10 min and quick chill on ice. Collect thecontents of the tube by brief centrifugation and add:4 ul 5X First Strand Buffer 2 ul 0.1 M DTT1 ul 10 mM dNTP Mix (10 mM each dATP, dGTP, dCTP and dTTP at neutral pH)Mix contents of the tube gently and incubate at 42°C for 2 min. Add 1ul (200 units) of SUPERSCRIPT II, mix by pipetting gently up and down.Incubate 50 min at 42°C. Inactivate the reaction by heating at 70°C for15 min. The cDNA can now be used as a template for amplification inPCR. However, amplification of some PCR targets (those >1 kb) mayrequire the removal of RNA complementary to the cDNA. To remove RNAcomplementary to the cDNA, add 1 ul (2 units) of E. coli RNase H andincubate 37°C for 20 min.*Alternatively 50-250 ng of random primers or 2 pmole of a gene specificprimer may be used. Use of random primers requires incubation at 25°Cfor 10 min before the 42°C incubation.IV. STORAGE AND STABILITYThe enzyme, buffer and the 0.1 M DTT should be stored at -20°C. Avoid-70°C! Enzyme is stable at least 12 months at -20°C and 3 days at 0°C.V. NOTESRELATIVE RESULTS USING 2 ug OF A 7.5 KB RNA (40% which is poly A+) mMLV H+ SuperScript I SuperScript IITotal cDNA 381 ng 586 ng 631 ngFull Length cDNA 181 ng 292 ng 355 ngSee also FAQs for Reverse Transcriptase PCR (RT PCR) FAQs for DNA, RNA Modifying Enzymes VI. REFERENCES1. Technical Bulletin 18064-1.2. Gerard,G., Schmidt,B., Kotewicz,M., Campbell,J.(1992) Focus 14:3, 91.cDNA Synthesis by Moloney Murine Leukemia Virus RNase H-Minus Reverse Transcriptase Possessing Full DNA Polymerase Activiity3. Mangan,J., Butcher,P.(1993) Focus 15:3, 67.Running AMOC with SUPERSCRIPT II Reverse Transcriptase VII. RELATED PRODUCTSPCR SUPERMIXTaq DNA Polymerase, RecombinantRibonuclease H
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