Synthesis of Double-Stranded cDNA from Purified Poly(A) mRNA

 

Use T7-(dT)24 oligomer for priming first-strand cDNA synthesis:

 

5-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3

 

High-quality HPLC-purifed T7-(dT)24 primer should be used (NOT PAGE-purified primer). This is available from GENSET.

 

1st strand synthesis

starting material: 0.2-5mg poly (A) mRNA

determine correct volumes of H2O and reverse transcriptase to be used: for every mg of RNA, need 1ml of SSIIRT (200U/ml). For mRNA quantity < or = 1mg, use 1ml of SSII RT.

 

Procedure:

  1. To 1.5mL RNase-free polypropylene tube, add:

Volume final concentration

    1. T7-(dT)24 primer 1ml 100pmol
    2. mRNA 0.2 to 5 mg
    3. DEPC-treated H2O enough to bring final volume (after step3) to 20ml

2.Add to tube:

a. 5X first strand buffer 4ml 1X

b. 0.1M DTT 2ml 10mM

c.10mM dATP, dCTP,

dGTP, dTTP 1ml 500mM each

  1. Add to tube:
    1. Superscript reverse

transcriptase 1ml per mg mRNA 200U to1000U

 

FINAL VOLUME FOR REACTION: 20ml

 

Second Strand cDNA synthesis

  1. Place first strand reactions on ice. Centrifuge briefly to bring down condensation on sides of tube.
  2. Add to the first strand synthesis tube the reagents listed below:

 

DEPC-treated water 91ml

5X second strand reaction buffer 30ml 1x

10mM dATP, dCTP, dGTP, dTTP 3ml 200mM each

10U/ml DNA ligase 1ml 10U

10U/ml DNA polymeraseI 4ml 40U

2U/ml RNase H 1ml 2U

 

  1. Gently tap tube to mix, spin to remove condensation and incubate at 16C for 2 hrs in a cooling waterbath.
  2. Add 2ml [10U] T4 DNA polymerase
  3. Cool for 5 minutes at 16C
  4. Add 10ml 0.5M EDTA
  5. Proceed to cleanup DNA or store at -20C.