T cleavage reaction mix

Unincorporated dNTPs in the amplification products were dephosphorylated by adding 1.7 μL Dnase free water and 0.3 μL (0.5 U) shrimp alkaline phosphatase (SAP) (Sequenom, Inc., San Diego, CA, USA). Each reaction was incubated at 37 °C for 40 min, and SAP was then heat inactivated for 5 min at 85 °C. Subsequently, samples were incubated for 3 h at 37 °C with 5 μL of T‐cleavage reaction mix (Sequenom), containing 3.21 μL RNAse‐free water, 0.89 μL 5× T7 polymerase buffer, 0.22 μL T‐cleavage mix, 0.22 μL 100 mm DTT, 0.40 μL T7 RNA & DNA polymerase and 0.06 μL RNAse A, for concurrent in vitro transcription and base‐specific cleavage. The samples of cleaved fragments were then diluted with 20 μL water. Conditioning of the cleavage reaction was carried out by adding 6 mg of clean resin.

Unincorporated dNTPs in the amplification products were dephosphorylated by adding 1.7 μl DNase free water and 0.3 μl (0.5 U) shrimp alkaline phosphatase (SAP) (Sequenom, Inc., San Diego, CA, USA). Each reaction was incubated at 37 °C for 40 minutes, and SAP was then heat inactivated at 85 °C for 5 minutes. Subsequently, samples were incubated at 37 °C for 3 hours with 5 μl of T-cleavage reaction mix (Sequenom), containing 3.21 μl RNAse-free water, 0.89 μl 5X T7 polymerase buffer, 0.22 μl T-cleavage mix, 0.22 μl 100 mM DTT, 0.40 μl T7 RNA polymerase and 0.06 μl RNase A, for concurrent in vitro transcription and base-specific cleavage. The samples of cleaved fragments were then diluted with 20 μl water. Conditioning of the cleavage reaction was carried out by adding 6 mg of clean resin.

Unincorporated dNTPs in the amplification products were dephosphorylated by adding 1.7 μL DNase free water and 0.3 μL (0.5 U) shrimp alkaline phosphatase (SAP) (Sequenom, Inc., San Diego, CA, USA). Each reaction was incubated at 37 °C for 40 min, and SAP was then heat inactivated at 85 °C for 5 min. Subsequently, samples were incubated at 37 °C for 3 h with 5 μL of T-cleavage reaction mix (Sequenom), containing 3.21 μL RNAse-free water, 0.89 μL 5X T7 polymerase buffer, 0.22 μL T-cleavage mix, 0.22 μL 100 mM DTT, 0.40 μL T7 RNA polymerase and 0.06 μL RNase A, for concurrent in vitro transcription and base-specific cleavage. The samples of cleaved fragments were then diluted with 20 μL of water. Conditioning of the cleavage reaction was carried out by adding 6 mg of clean resin.