Please read the excellent descriptions of handling RNA and of cDNA 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 µg poly-A RNA
50 µCi (5 µl) alpha-32-P-dCTP
Add to the RNA:
5 µl H20
0.5 µl 100mM Me(Hg)OH [in fume hood, DANGEROUS]
Incubate at room temperature for 10 min. Then add:
1 µl 700mM B-mercaptoethanol (BME) diluted in H2O
0.5 µl RNAsin (15units; stored at -30°C)
Incubate at room temperature for 5 min. Then add:
5 µl oligo-dT (@1mg/ml)
2.5 µl 1M Tris-HCl, pH 8.3 (pH at 42°C)
2 µl reverse transcriptase @ 19U/µl (higher than Maniatis)
Incubate at 42°C, 3 hr. Add 1µl of 0.5 M EDTA to stop the reaction. Extract with 15µl phenol 15µl CHCl3.
Determine the efficiency of first strand cDNA synthesis:
Count:
- 1µl of the reaction mixture spotted on a GF/C filter. This will measure the total radioactivity in the sample.
- 1µl of the reaction mixture plus 5 µl 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. Wash 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 µg of cDNA.
- Moles of dCTP = "a" =
(% incorporated above)(molar concentration of dCTP)
where the molar concentration = cold dCTP concentration since 32P-labeled dCTP is just a chase).
- Labeled moles expected with 100% efficiency = b =
(about 25% of each base)(µg RNA)/(about 330 g/mole)
- % Efficiency = a/b x 100
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.
Cleave 5' Overhanging Ends:
5 µl (1 µg) ds-cDNA [use only 1/2 of 2µg; store rest at -20°C]
2 µl 10X T4 polymerase buffer (per Maniatis)
12 µl H2O
4 µl dNTPs (1µl each of 2 mM stock)
0.5 µl T4 DNA polymerase (6U/µl stock; 3U)
Incubate 5 min, 37°C. Add 1 µl 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 µg ds-DNA
100 mM Tris-HCl, pH 8.0
10 mM EDTA
5 µM S-adenosyl-methionine
1 U EcoRI methylase
Incubate 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 µg ds-cDNA, dry
2 µl linker (1µg at 0.5µg/µl)
1 µl 10X ligation buffer
1 µl 100 mM DTT
1 µl 10 mM ATP
4 µl H2O
1 µl T4 DNA ligase (1 U/µl stock)
Incubate at 15°C overnight. Add 1 µl of 0.5M EDTA and µl of TE. Extract once with phenol:CHCl3 and then precipitate once with ammonium acetate/ethanol. Dry the DNA.
EcoRI Digest:
1 µg ds-cDNA
22.5 µl 0.1X TE
2.5 µl 10X RI buffer
0.25 µl 100 mM DTT
2 µl EcoRI (20 U from a 10 U/µl stock)
Incubate 4 - 5 hrs, 37°C, then add 10 U EcoR I and incubate an additional hr at 37°C. Add 2 µl 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 6µl of 0.1X TE for ligation.
Ligate cDNA to Vector:
6 µl cDNA (1 µg)
2.2 µl gt11 (1 µg)
1 µl 10X ligation buffer
1 µl T4 ligase (1 Unit @ 1 U/µl)
Incubate overnight at 15°C, package and plate, or transform, and then screen. Using this protocol, we obtained about 100,000 pfu per µg of packaged DNA).
Notes on Reagents
- Me(Hg)OH
- DANGEROUS, store in fume hood
- RNAsin
- RNAse inhibitor (Promega Biotek, 30 U/µl)
- 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. coli
- E. coli DNA Ligase
- For second strand synthesis, specified by Okayama and Berg (New England Biolabs)
- T4 DNA Polymerase
- Used to cleave 3' overhanging ends
- EcoRI Linker
- Phosphorylated; Collaborative Research
- T4 DNA Ligase
- from IBI
Special thanks to Chris Beard in Dr. Jerry Manning's laboratory for many useful comments on cDNA synthesis, August 1985.
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