Please read the excellent descriptions of handling RNA and of cD NA synthesis by J. Sambrook, E. F. Fritsch, and T.Maniatis in Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, Second Edition, New York, 1989). The methodology for the synthesis of the first strand of the cDNA summarized below is based primarily on this source.1st Strand cDNA Synthesis Dry in a vacuum desiccator: 5 g poly-A RNAnb sp; 50 Ci (5 l) alpha-32-P-dCTP Add to the RNA: 5 l H20 0.5 l 100mM Me(Hg)OH [in fume hood, DANGEROUS] Incubate at ro om temperature for 10 min. Then add: 1 l 700mM B-mercaptoethanol (BME) diluted in H2O 0.5 l RNAsin (15units; stored at -30°C) Incubate at room temperature for 5 min. Then add: 5 l oligo-dT (@1mg/ml) 2.5 l 1M Tris-HCl, pH 8.3 (pH at 42°C) 4 l 1M KCl (optimum final concentration of 140-150mM is critical) 1 l 250 mM MgCl2 2.5 l 10 mM dTTP 2.5 l 10 mM dATP 2.5 l 10 mM dGTP 2.5 l 10 mM dCTP (instead of Maniatis\'\' 5mM)2 l reverse transcriptase @ 19U/ l (higher than Maniatis)Incubate at 42°C, 3 hr. Add 1 l of 0.5 M EDTA to stop the reaction. Extract with 15 l ph enol + 15 l CHCl3.Determine the efficiency of first strand cDNA synthesis:Count: 1 l of the reaction mixture spotted on a GF/C filter. This will measure the total radioactivity in the sample.1 l of the reaction mixture plus 5 l of a 40 mg/ml yeast tRNA stock (dissolved in water) and 5 ml of 10% TCA. Incubate on ice, 15 min. Filter through a GF/C filter. W ash the filter 4 times with 5 ml of cold 10% TCA, followed by 5 ml of 95% ethanol. This will measure the radioactivity incorporated into high molecular weight nucleic acids. Use the following to calculate the total g of cDNA. Moles of dCTP = \"a\" =(% incorporated above)(molar concentration of dCTP)where the molar concentration = cold dCTP concentration since 32P-la beled dCTP is just a chase).Labeled moles expected with 100% efficiency = b =(about 25% of each base)( g RNA)/(about 330 g/mole) % Efficiency = a/b x 100 A good cDNA synthesis will contain up to 60-70% of the g of RNA (although this proportion can be quite variable). For example, 45% efficiency would produce 2.2 g of ss-cDNA from 5 g RNA, (representing 4.4 g of ds-cDNA).Ethanol precipitate with ammoniu m acetate. 35 l RNA35 l 4 M ammonium acetate (filter sterilized) 140 l absolute ethanolFreeze to solid white pellet on dry ice. Thaw, centrifuge at room temperature for 10 min in a high speed microcentrifuge. Resuspend in 35 l of 0.1X TE. Repeat the precipitation. Wash the pellet in 80% ethanol. Centrifuge for 10 min, and dry the pellet. Resuspend the final pellet in 4 0 l of 0.1X TE. Second Strand cDNA Synthesis (as per J. Gubler B. Hoffman, Gene 25:263, 1983; after H. Okayama P. Berg, Mol. Cell. Biol. 2:161, 1982). Mix the following components: 122 l Assuming you have about a 4 g equivalent of ds-DNA, use half of it. Store the remainder at -70°C for future use. Incubate the reaction components for 60 min at at 12°C, then 60 min at 22°C (on the bench). Add 8 l of 0.5M EDTA to stop the reaction, then phenol extract twice. Precipitate the DNA twice with ammonium acetate /ethanol. Wash in 80% ethanol, dry resuspend the ds-DNA in 10 l of 0.1X TE. Cleave 5\'\' Overhanging Ends:5 l (1 g) ds-cDNA [use only 1/2 of 2 g; store rest at -20°C] 2 l 10X T4 polymerase buffer (per Maniatis) 12 l H2O 4 l dNTPs (1 l each of 2 mM stock) 0.5 l T4 DNA polymerase (6U/ l stock; 3U) Incubate 5 min, 37°C. Add 1 l of 0.5M EDTA. Extract once with phenol:CHCl3. Precipitate once with ammonium acetate/ethanol, wash with 80% ethanol, and dry the pellet. Methylate to Protect EcoRI Sites in cDNA : This is an optional step if you are adding EcoR I linkers to a cDNA that may contain internal EcoR I site(s). Omit this step if there is no EcoR I site in the insert you are studying. The basis for this protocol was published by Greene, et al (J. Mol. Biol. 99:237-261, 1975), and by T. Maniatis et. al, (Cell 15:687, 1978). 1-2 g ds-DNA100 mM Tris-HCl, pH 8.0 10 mM EDTA5 M S-adenosyl-methionine1 U EcoRI methylaseIncubate for 1 hr at 37°C, then phenol extract. (If you are methylating oligonucleotides, incubate at 12°C), Precipitate in ammonium acetate/ethanol. Wash with 80% ethanol, and dry the DNA. See Greene, et al (cited above) for assay to monitor methylation. Ligate to Linkers:1 g ds-cDNA, dry2 l lin ker (1 g at 0.5 g/ l)1 l 10X ligation buffer1 l 100 mM DTT1 l 10 mM ATP4 l H2O1 l T4 DNA ligase (1 U/ l stock)Incubate at 15°C overnight. Add 1 l of 0.5M EDTA and l of TE. Extract once with phenol:CHCl3 and then precipitate once with ammonium acetate/ethanol. Dry the DNA. EcoRI Digest:1 g ds-cDNA22.5 l 0.1X TE2.5 l 10X RI buffer0.25 l 100 mM DTT2 l EcoRI (20 U from a 10 U/ l stock)Incubate 4 - 5 hrs, 37°C, then add 10 U EcoR I and incubate an additional hr at 37°C. Add 2 l of 0.5M EDTA, extract once with phenol:CHCl3, and precipitate twice with ammonium acetate/ethanol. Wash the pellet with 80% ethanol. Dry the DNA. Resuspend in 6 l of 0.1X TE for ligation. Ligate cDNA to Vector:6 l cDNA (1 g)2.2 l gt11 (1 g)1 l 10X ligation buffer 1 l T4 ligase (1 Unit @ 1 U/ l)Incubate overnight at 15°C, package and plate, or transform, and then screen. Using this protocol, we obtained about 100,000 pfu per g of packaged DNA). Notes on Reagents Me(Hg)OH DANGEROUS, store in fume hoodRNAsin RNAse inhibitor (Promega Biotek, 30 U/ l)Oligo-dT Primer for first strand (Collaborative Research, dT 12-18; stock 1 mg/ml in H2O; store -20°C or -70°C)RTase Reverse transcriptase, store at -70°C (Molecular Genetics Resources, Tampa,Florida); we have also used Life Sciences RTase and obtained about 10% first-strand synthesis efficiency at 140mM KCl, and a bit lower at 80 mM KCl. BNAD beta-NAD (Sigma, mw=663.4)RNAseH from E. coli (Boehringer Mannheim Biochemicals)DNA Pol-I DNA polymerase I from E. coliE. coli DNA Ligase For second strand synthesis, specified by Okayama and Berg (New England Biolabs)T4 DNA Polymerase Used to cleave 3\'\' overhanging endsEcoRI Linker Phosphorylated; Collaborative ResearchT4 DNA Ligase from IBI