Phosphatase buffer

RNase H (TaKaRa) was added to the obtained reverse-transcribed products to degrade free RNA. To synthesize double-strand cDNA (dscDNA), anchored random primers were added and incubated at 65°C for 5 min and then placed on ice for 5 min for denaturation (Hameed et al., 2020 (link)). After this, 1 μL of Klenow fragment (TaKaRa), 1 μL of 10 mM dNTPs (TaKaRa), 2 μL of 10 × Klenow buffer (TaKaRa), and 6 μL of ddH₂O (TaKaRa) were added and incubated at 37°C for 60 min, followed by an incubation at 75°C for 10 min. To remove phosphates and the free single-strand nucleic acid in the dscDNA reaction, 0.5 μL of Exonuclease I (TaKaRa), 1 μL of alkaline phosphatase (TaKaRa), 5 μL of 10 × phosphatase buffer (TaKaRa), and 24 μL of DEPC H₂O (TaKaRa) were added and incubated at 37°C for 60 min, followed by an incubation at 75°C for 10 min.

Before the synthesis of dscDNA, 1 μL of RNase H (TaKaRa) was added to the obtained products to degrade the free RNA. To synthesize dscDNA, anchored random primers were added and incubated 75°C for 5 min and then on ice for 5 min for denaturation. Then, 1 μL of Klenow fragment (TaKaRa), 1 μL of 10 mM dNTPs (TaKaRa), 2 μL of 10 × Klenow buffer (TaKaRa), and 6 μL of dd H₂O (TaKaRa) were added and the samples were incubated at 37°C for 60 min, followed by 75°C for 10 min. Then, 0.5 μL of exonuclease I (TaKaRa), 1 μL of shrimp alkaline phosphatase (SAP, TaKaRa, Dalian, China), 5 μL of 10 × phosphatase buffer (TaKaRa), and 24 μL of DEPC H₂O (TaKaRa) were added and incubated at 37°C for 60 min followed by 75°C for 10 min to eliminate the phosphates and the free single-strand nucleic acid in the dscDNA reaction.