Subsequently, the reverse transcriptase reaction was carried out in 40 μl reaction volume containing 0.5 mM dNTPs, 2.5 ng/μl random hexamer primers, 5 mM MgCl2, 0.01 M DTT, 2 μl RNase-out, 2.5 U/μl Superscript II Reverse Transcriptase (Invitrogen, Breda, The Netherlands), and 1× reaction buffer supplied by the manufacturer of the enzyme (Invitrogen).
All ctDNA markers were amplified with NEB Taq using the buffer supplied by the manufacturer (NEB co., Japan).
The amplification reaction (25 μl) contained 50 ng DNA, 0.50 μM of each primer, 250 μM dNTPs, 1.5 mM MgCl2, and 1.25 U pfu polymerase in the buffer supplied by the manufacturer.
PCR reactions were performed in 50 μl volumes in solutions containing 1 μl aliquots of the respective cDNA reaction mixture, 0.2 μM of gene-specific primers, 10 mM dNTPs, one unit of Taq DNA polymerase (Invitrogen), and 10× Taq buffer supplied by the manufacturer.
For the first-strand cDNA synthesis, 100 ng of mRNAs from the same RNA samples used for the microarray experiments was reverse-transcribed in a total volume of 50 μl that contained 10 ng of oligo (dT 12 18 primer, 2.5 mM dNTP, and 100 units of SuperScript™ III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) in reaction buffer supplied by the manufacturer.
One ITS region was amplified with primers 16S-F (5'- andTCGGTGGAGTAACCGT-3') and 23S-R (5'- TGTTAGTCCCGTCCTTCAT-3'). PCR reactions (25 μl) contained 50 ng DNA, 0.5 μM of each primer, 250 μM dNTPs, and 2.5 U Taq DNA polymerase in the buffer supplied by the manufacturer.
After this incubation, the contents of the plate were discarded and the plate was rinsed 5 times with wash buffer supplied by the manufacturer.
Unincorporated primers and nucleotides were removed using the S.N.A.P.™ Column purification system according to the manufacturer's protocol (Invitrogen) and the purified amino allyl-modified cDNA was re-suspended in 5 µl of the coupling buffer supplied by the manufacturer.
PCR was performed with Ampli Taq or Ampli Taq Gold DNA polymerase (Applied Biosystems, Foster City, CA) with buffers supplied by the manufacturer, at a 2.5 mM MgCl2 concentration.
The duplexes are incubated with M.SssI (10 and 25 fold excess) in the buffer supplied by NEB, supplemented with 300 µM of AdoMet and 100 µg/mL of BSA.
5' [γ32P] labeled duplexes ds-X (for MSss.I) (0.05 pmol) were incubated with or without M.SssI (1 µL of M.SssI (ca. 0.1 mg/mL)) in the buffer supplied by NEB supplemented with 1 µM of AdoMet and 100 µg/mL of bovine serum albumin (BSA).
