Rna ligase 2 buffer

In the first
ligation reaction, the splint adapter to tRNA molar ratio was 1:1.25.
The tRNA sample is first heated to 95 °C for 2 min, allowed to
cool for 2 min, then placed on ice for 2 min. The reaction was carried
out at room temperature in a DNA LoBind tube for 45 min. Its constituents
were 1× RNA ligase 2 buffer (NEB) supplemented with 5% PEG 8000,
2 mM ATP, 6.25 mM DTT, 6.25 mM MgCl₂, and 0.5 units/μL
T4 RNA ligase 2 (10,000 units/mL). For single tRNA isotype libraries
(for tRNA^fMet, tRNA^Lys, or tRNA^Phe), 16 pmol of splint adapter and 20 pmol of tRNA (∼500 ng),
in a total reaction volume of 20 μL, were used. The tRNAs for
these libraries have ACCA 3′ termini. The splint adapter used
was the form with a UGGU overhang. Total tRNA reactions using all
four splint adapters were performed using 32 pmol of adapter (8 pmol
of each of the four adapters) and 40 pmol (∼1 μg) of
total tRNA in a reaction volume of 40 μL. For total tRNA runs
using only one of the four adapters, 16 pmol of adapter and 20 pmol
(∼500 ng) of total tRNA were used in a reaction volume of 20
μL.

All ligases and buffers were obtained from New England Biolabs, Inc (NEB), Ipswich, MA, USA. The concentrations of the selling stocks of the enzymes were: T4 DNA Ligase high concentration (45 pmol/μl), T4 RNA Ligase 2 (6.6 pmol/μl) and SplintR Ligase (10.5 pmol/μl). To compare ligation activites, 10 pmol of enzyme was used in each ligation reaction according to the manufactures protocol. T4 DNA Ligase and SplintR Ligase reactions were performed in 1× T4 DNA Ligase buffer. This buffer has the same composition as SplintR reaction buffer (NEB). T4 RNA Ligase 2 reactions were performed in 1X RNA Ligase 2 buffer (NEB). The microRNA reverse transcription kit and TaqMan universal PCR master mix were purchased from Life Technologies (Carlsbad, CA, USA). The One Taq Hot Start 2X PCR Master Mix was obtained from NEB.