cDNA

PRODUCTION OF cDNA

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 µg poly-A RNA
&nb sp;        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 ro om 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)
          4 µl 1M KCl (optimum final concentration of 140-150mM is critical)
          1 µl 250 mM MgCl2
          2.5 µl 10 mM dTTP
          2.5 µl 10 mM dATP
          2.5 µl 10 mM dGTP
          2.5 µl 10 mM dCTP (instead of Maniatis' 5mM)
          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 ph enol + 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. 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 µg 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)(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 ammoniu m 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 4 0 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 1;g/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< /FONT>	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 lin ker (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

Special thanks to Chris Beard in Dr. Jerry Manning's laboratory for many useful comments on cDNA synthesis, August 1985.

Send comments and updates to Dr. Bart Frank, Arthritis and Immunology Program, OMRF

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Copyright 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Production of cDNA. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/cdna.html). 1997. Oklahoma City. Revision Date: October 2, 199 7."