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

 

àUse 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

àdetermine 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 16°C for 2 hrs in a cooling waterbath.
  2. Add 2ml [10U] T4 DNA polymerase
  3. Cool for 5 minutes at 16°C
  4. Add 10ml 0.5M EDTA
  5. Proceed to cleanup DNA or store at -20°C.