A good cDNA synthesis will contain up to 60-70% of the µg of RNA (although this proportion can be quite variable). For example, 45% efficiency would produce 2.2µg of ss-cDNA from 5 µg RNA, (representing 4.4 µg of ds-cDNA).
Ethanol precipitate with ammonium acetate.
35µl RNA
35µl 4 M ammonium acetate (filter sterilized)
140µl absolute ethanol
Freeze to solid white pellet on dry ice. Thaw, centrifuge at room temperature for 10 min in a high speed microcentrifuge. Resuspend in 35 µl 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 40 µl 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:
| Final Concentration | µl used for 2µg DNA | Stock Concentration |
|---|---|---|
| ds-cDNA | 20 µl | |
| 20 mM Tris-HCl 7.5 | 4 µl | 1 M |
| 5 mM MgCl2 | 10 µl | 0.1 M |
| 10 mM (NH4)2S04 | 2 µl | 1 M |
| 100 mM KCl | 20 µl | 1 M |
| 0.15 mM BNAD | 2 µl | 15 mM |
| 50 µg/ml BSA | 5 µl | 2 mg/ml |
| 40 µM dATP | 0.8 µl | 10mM |
| 40 µM dCTP | 0.8 µl | 10mM |
| 40 µM dTTP | 0.8 µl | 10mM |
| 40 µM dGTP | 0.8 µl | 10mM |
| 8.5 U/ml E.coli RNAseH | 1.9 µl | 0.9U/µl |
| 230 U/ml DNA polymerase-I | 9.2 µl | 5U/µl |
| 10 U/ml E.coli ligase | 0.4 µl | 5U/µl |
| H20 | 122µl |
Assuming you have about a 4 µg 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 µl 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 10µl of 0.1X TE.
