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œœœœœ@DS cDNA synthesis@œœœœœ



@The method described here is for the synthesis of blunt-ended double-stranded cDNA, suitable for cloning either by blunt-end ligation or linker-ligation into a plasmid or M13 vector. The most important factor in synthesizing cDNA is that the mRNA template be as pure as possible - free of RNase, rRNA, inhibitory compounds (especially polysaccharides), and, if a specific cDNA is desired, extraineous poly(A)-containing RNAs. This is a technically difficult technique, so don't be discouraged if it doesn't work the first couple of times. If it doesn't work, retest the poly(A)-containing RNA by agarose-urea electrophoresis to see if it is still intact.
@Strict RNase-free technique should be used throughout this procedure. All glassware, plasticware, and buffers should be autoclaved, except for the dNTPs, 2-ME, and oligo(dT)12-18.

  1. NOTES

    * If 32P and 3H is needed only to trace the reaction, the isotopes can be decreased to 5uCi each.

    * If you desire or need to use an RNase inhibitor, then replace the 2-ME with 2.5ul 0.1M DTT and add 5 units RNasin and 100ug/ml BSA during the first strand synthesis.

  2. Reagents

    1. 10X cDNA buffer (per 100ml)
      final conc
      6.06 grams Tris-OH 0.5M
      2.03 grams MgCl2-7H2O 0.1M
      10.44 grams KCl 1.4M
      @Adjust to pH8.3 at 42C with conc. HCl

    2. 0.3M 2-mercaptoethanol
      @131ul 2-ME plus 4.87ml ddH2O

    3. 20mM dNTPs
      ddH2O
      dATP 25mg 2.15ml
      dCTP 25mg 2.35ml
      dTTP 25mg 2.23ml
      dGTP 25mg 2.12ml
      Add ddH2O directly to the bottle, dissolve, and adjust to neutrality with 50mM Tris, pH7

    4. dNTP mix - mix 5ul of each 20mM dNTP just before starting.

    5. TEN (per liter)
      final conc.
      1.2114 grams Tris-OH, pH 7.8 10mM
      0.5844 grams NaCl 10mM
      0.3802 grams Na4EDTA 1mM

    6. 8u/ml oligo(dT)12-18

    7. Add 125ul ddH2O/A260 unit directly to the vial

    8. 1M HEPES
      @pH 6.9 - 5.206 grams per 20ml

    9. S1 nuclease buffer (per 100ml)
      final conc.
      0.4082 grams Na acetate 30mM
      1.7532 grams NaCl 0.3M
      0.0409 grams ZnCl2 3mM

    10. 2.5M Na acetate
      @34.02 grams per 100ml

    11. 25:1 chloroform/1-octanol
      @10ml 1-octanol + 240ml chloroform

  3. Mixture

    1. First strand final reaction mixture:
      50mM Tris, pH 8.3 (at 42C)
      10mM MgCl2
      140mM KCl
      30mM 2-ME
      0.04 A260 units oligo(dT)12-18
      1mM @ dATP, dCTP, dGTP, and dTTP
      50uCi 32P-dCTP
      5ug poly(A)-containing RNA
      50 units reverse transcriptase
      50ul total volume @

    2. Second strand final reaction mixture:
      0.1M HEPES, pH 6.9
      0.75mM @ dATP,dCTP, dGTP, and dTTP
      50uCi @ 3H-dCTP and 32P-dCTP
      70mM KCl
      100 units DNA polymerase I
      5mM MgCl2
      15mM 2-ME
      @ other sscDNA syn. leftover
      100ul total volume @

  4. Methods

    1. First strand synthesis:

      1. Transfer 50uCi a32P-dCTP to a 500ul microfuge tube, plug it with cotton, and cover it with a double layer of parafilm. Puncture the parafilm several times with a needle.
      2. Dry in a vacuum dessicator under vacuum to complete dryness (usually 15-30 min.). Remove and discard the parafilm and cotton.
      3. Working on ice, add the following reagents to the vial:
        5ul 10X cDNA buffer
        5ul 0.3M 2-ME
        5ul 8u/ml oligo(dT)12-18
        10ul dNTP mix
        5ul 1ug/ul poly(A)-containing RNA
        12.75ul ddH2O

      4. Vortex, and add 7.25ul reverse transcriptase (50 units). GENTLY MIX, spin 5 sec., and incubate for 1 hr at 42C.
        Drop in a boiling water bath for 2 min., and rapidly cool on ice. Remove a 2.5ul aliquot for later assaying.

    2. Second strand synthesis:

      1. Centrifuge the remainder for 1 min., and transfer the liquid (47ul) to a 500ul microfuge tube containing 5uCi 3H-dCTP dried down as described in steps 1 & 2.
      2. Add 10ul 1M HEPES, pH 6.9, 5ul dNTP mix, and 34ul ddH2O to the vial, and vortex. Then add 13ul DNA polymerase I (100 units), gently mix again, and incubate at 15C for 3 hr.
      3. While the reaction mix is incubating, assay the sscDNA synthesis as follows:

        1. Dilute a 2.5ul aliquot of sscDNA with 40ul TEN.
        2. Add 5ul glycerol, and vortex.
        3. Apply this sample to a 0.5 x 30cm sephadex G50(50-150) column, equilibrated with TEN.
        4. Collect 32 @ 25 drop (0.5ml) fractions into 6ml scintillation vials containing 5ml scintillation fluid each.
        5. Cap and mix the vials, and count them by scintillation spectrophotometry.

      4. After incubating, add 10ul glycerol and 110ul 25:1 chloroform/1-octanol, and vortex. Centrifuge 1 min. to separate the phases.
      5. Using a pipette gun, collect the upper (aqueous) phase, being very careful not to get any of the lower (organic) phase. Apply the entire aqueous phase to a 0.5 x 30 cm Sephadex G50(50-150) column (equilibrated and run in TEN.).
      6. Run the column, and collect 36 @ 25 drop (0.5ml) fractions. Transfer a 25ul aliquot from each fraction into separate 6ml scintillation vials containing 5ml of scintillation fluid. Count each fraction using separate 32P and 3H windows.
      7. Pool the fractions containing the cDNA peak, and add 1/10 volume of 2.5M Na acetate and 2 volumes of 2-PrOH. Store overnight at -70C.

    3. S1 nuclease treatment:

      1. Pellet the dscDNA by centrifuging for 1 hr at 35KRPM (SW55Ti rotor) at 4C. Discard the supernatant, allow the excess fluid to drain off, and dry the pellet under vacuum.
      2. Dissolve the invisible pellet by adding 520ul S1 nuclease buffer and a baby stir bar (autoclaved). Stir very slowly overnight at 4C.
      3. Transfer the solution to a 1.5ml microfuge tube, and spin 1 min. to remove junk.
      4. Transfer to a fresh 1.5ml microfuge tube, then remove a 25ul 'Pre-S1 nuclease' aliquot for later analysis (store at -20C). Add 6ul S1 nuclease (60 units) and incubate at 37C for 1 hr.
      5. Remove a 25ul 'Post-S1 nuclease' aliquot for later analysis (store at -20C). Add 500ul 25:1 chloroform/1-octanol and vortex.
      6. Centrifuge for 1 min. to separate the phases. Carefully collect the upper (aqueous) phase, and transfer it to a fresh 1.5ml microfuge tube. Add 1ml 2-PrOH, mix, and store overnight at -70C.
      7. Pellet the dscDNA for 1 hr, and decant the supernatant. Allow the pellet to drain dry, the dry under vacuum. Store at 4C until use.
      8. Treat the pre- and post- S1 nuclease samples exactly as described in step 2, but count the 3H and 32P channels separately.


Rosemarie Spencer (personal communication)
Buell, et al. 1978 J. Biol. Chem. 253:2471-2482
Wickens, et al. 1978 J. Biol. Chem. 253:2483-2495
Land, et al. 1981 Nucl. Acids Res. 9(10):2251
J. Vaughn (personal communication)
BRL Product Profile: AMV Reverse transcriptase
Monahan, et al. 1976 Biochemistry 15:223-233
Baulcombe and Verma 1978 Nucl. Acids Res. 5(11):4141

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