Step 1: Preparation of double-stranded oligonucleotide substrates
Annealed nicked substrates are suitable for ligation by SplintR Ligase. During development of the product we used an acceptor DNA oligonucleotide, a 5'-phosphorylated, 3'- 6-carboxyfluorescein (FAM)-labeled DNA donor oligonucleotide, and an RNA splint of complementary sequence. Individual oligonucleotides were purchased from a commercial DNA synthesis vendor with the required modifications and in HPLC-purified form. The oligos were resuspended in milli-Q H2O to a concentration of 100 µM and diluted as needed. The FAM-labeled DNA donor (10 µM) was combined with 1.1 molar equivalents each of the DNA acceptor and the DNA or RNA splint in oligonucleotide annealing buffer. This mixture was heated to 85°C for 2 minutes and allowed to cool slowly in a heat block to room temperature over 3 hours. Annealed DNA/RNA hybrid stocks (10 µM in the FAM-labeled fragment) can be used immediately for a ligation reaction or, alternatively, can be stored at −20°C. Working stocks were prepared by diluting the substrates to 1 µM.
Step 2: SplintR Ligation Assay
- Reactions can be performed in typical plasticware (microfuge tubes, PCR plates, etc).
- Thaw reagents and keep on ice.
- Add components to a final volume appropriate for downstream steps (20 µl for this example):
|10X Reaction Buffer
|Substrate (1 µM)
||1 µl (25U)
Note: In this example the substrate is present at 100 nM, and ligase at 500 nM. We recommend a starting concentration of SplintR around 2-fold excess over ligatable ends. See the product datacard or website for more detailed guidance on reaction conditions, diluents, etc.
- Incubate for 15-60 minutes at 25°C.
- Stop reaction using either heat inactivation at 65°C or other method appropriate for downstream
steps such as quenching with EDTA.
- Keep reaction on ice until needed (hrs) or store at -20°C.
During development, our nick ligation assays were quenched in 50 mM EDTA, 0.1% Triton-X, diluted with water so that the total concentration of FAM-labeled molecules was ~1 nM, and analyzed by capillary electrophoresis (CE) fragment analysis on an Applied Biosystems 3730xl Genetic Analyzer (96 capillary array). For reactions run at < 50 nM substrate, the diluted samples were desalted using Edge-Bio PerformaTM VTR 96 well plates before injection. The concentration of each FAM-labeled species was determined as the area of the peak in question divided by the total area of all peaks in the FAM channel, multiplied by the initial concentration of substrate.