Generation of DIG-labeled cDNA

(Incorporation of Digoxigenin-11-dUTP during PCR)

Final concentration for nucleotides mix:

                                    dGTP                          2.0 mM

                                    dATP                          2.0 mM

                                    dCTP                          2.0 mM

                                    dTTP                           1.3 mM

                                    Dig-11-dUTP                     0.7 mM

NB: The use of DIG-11-dUTP, alkali labile is especially recommended when stripping and reprobing of Southern blots is planned.

1. Mix 1ml of Oligo (dT)_12-18 – primer (500mg/ml) and 1ml of nucleotides mix with 11ml of total RNA (appr. 15 to 20 mg).

2. Heat mixture to 70ºC for 10 min and quick chill on ice.

3. Centrifuge briefly the tube and add:

4ml 5X first strand buffer

2ml 0.1M DTT

1ml Superscript II (200U)

4. Incubate at 42ºC for 90 min.

5. The reaction is inactivated at 70ºC for 15 min.

6. RNA complementary to the cDNA is removed by adding 1ml of E. coli RNase H (2U) and incubating at 37ºC  for 20 min.

7.  The labeled probe can be stored for a long period at -20ºC . It's important to estimate the probe yield by the Direct Detection Procedure.