The oligomeric structure was characterized by size-exclusion chromatography using a Superdex 200 300/10 GL column (GE Healthcare) equilibrated with buffer B. The primers used to generate constructs for genetic manipulation were designed using the TcNMT X10/1 and flanking sequences in TriTrypDB as a template (Table 1).
Five microliters of RNA was combined with the appropriate primers and probes in buffer with the TaqMan RT-PCR ready-mix kit (PE Applied Biosystems, Foster City, CA).
The total RNA (1 μg) was subjected to the reverse transcription (RT) in 20 μl buffer with oligo- dT 15 primer using a RT system (Proligo- dT 15on, WI, USA).
The ligation was performed in a 1 × T4 DNA Ligase Buffer, with a Saturation Primer concentration of 10 uM and T4 DNA Ligase (2 U/mL), at 30°C for forty-five minutes on a rotisserie.
The forward primer described in Radtkey et al. [18] was used in combination with the primer MHCI-Int-R1 in a 25 µl PCR reaction with the following reagents and conditions: 2.5 U AmpliTaq DNA Polymerase (Applied Biosystems), 200 µM of each dNTP, 1 µM of each primer, 1.5 mM MgCl2, and 1× PCR Buffer without MgCl2.
Start with the primer brush.
Spray the area with the primer.
The first round was performed with the primers, Platinum Taq DNA Polymerase, 10X Reaction Buffer, MgCl2 (Invitrogen, USA) and deoxyribonucleotide triphosphates (dNTP; GE Healthcare, USA).
Approximately 2 µg of eluted bisulfite-treated DNA library had the complementary strand synthesized by primer extension in Platinum® Taq buffer with 10 pmol of primer P2-BtnB in the presence of 50 µM dNTPs, 2.5 mM MgCl2 and 1 U Platinum® Taq (Invitrogen), with temperature cycling of 3 min at 94°C before 15 min at 65°C, then cooling to 4°C.
For hybridization and ligation, 20 nM of the padlock probe is incubated in 1× phi29 buffer with 60 nM surface primer, 1 mM ATP, and 0.02 U/μL T4 DNA ligase.
