1. DNase treatment of total RNAs

On ice, mix the following:

*         RNAs                                        10ug (not more than 29.5 ul)

*         10 * RTase buffer                       5ul

*         RQ1 DNase                               2ul

*         H2O                                           up to 36.5ul

Mix well, spin down and incubate at 37C for 15min.

Inactive the DNase at 65C for 10min, then transfer the tubes on ice.

2. RTase (Synthesis of the first strand cDNAs)

On ice, add the following to the DNase-treated RNAs:

*         0.1M DTT                                5ul

*         10mM dNTPs                         5ul

*         0.5ug/ul oligo-dT                    2ul

*         MMLV-RTase                        1ul

*         RNase-inhibitor                      0.5ul

Incubate the tubes 1 hour at 37C then 2min at 92C.

For long storage, store the cDNAs at -80C, otherwise at -20C.

3. PCR

Perform the PCR by using 1 ul of cDNAs for a 50 ul PCR reaction.