Gcmelt buffer

Reverse transcription was performed from 1 μg of total RNA in a reaction volume of 20 μl, containing 0.4 mM Oligo(dT)₁₅ primer, 2 mM dNTP mix, 5 mM MgCl2, 20 U RNase inhibitor and 10 U AMV reverse transcriptase (AMV Reverse Transcriptase kit, Promega). The mixture was incubated 10 min at room temperature (RT), followed by incubations: one hour at 42°C, 5 min at 99°C and finally 10 min on ice. Then, the PCR was performed in a personal Robocycler Gradient 96 (Stratagene) in a reaction volume of 50 μl and comprised 2 μl of cDNA, 5 μl of 10X enzyme buffer, 10 μl of 5X GCmelt buffer (Ozyme), 200 nM of each primer, 0.4 mM of dNTP mix and 5 U Platinum Taq High fidelity DNA polymerase (Life Technologies). The program was applied as follows: 1 cycle of 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 54-57°C, 4 min at 68°C; 1 cycle of 10 min at 68°C. Following PCR, migration on agarose gel was performed to visualize the amplified fragment and check the size.

The TD-PCR was performed in a personal Mastercycler thermocycler (Eppendorf) in a reaction volume of 50 μl and comprised 100 ng gDNA or 2 μl of cDNA, 5 μl of 10X enzyme buffer, 10 μl of 5X GCmelt buffer (Ozyme), 200 nM of each primer, 0.4 mM of dNTP mix and 5 U Platinum Taq High fidelity DNA polymerase (Life Technologies). The program was applied as follows: 1 cycle of 5 min at 94°C; 9 cycles of 30 sec at 94°C, 30 sec at 64°C (−1°C per cycle), 1 min at 68°C; 30 cycles of 30 sec at 94°C, 30 sec at 55°C, 1 min at 68°C; and 1 cycle of 12 min at 68°C. Following PCR, migration on agarose gel was performed to visualize the amplified fragment and check the size.