- Produce 3 nmol of 5’-adenylated DNA or RNA oligos, triple the output of competitor kits.
- Avoid off-target effects like concatemerization and circularization seen in ligase-based adenylation methods.
- Adenylate oligos in a one-step, one-hour reaction instead of waiting weeks for purchased adenylated oligos.
- Scale up reactions as desired with no impact on adenylation efficiency.
Product Description:
The Adenylator™ DNA/RNA 5’-Adenylation Kit enables ligation-free,
efficient, 5’-end adenylation of 5’-monophosphorylated DNA and RNA
oligonucleotides. The kit provides a powerful, catalytic and
low-temperature alternative to 5’-adenylation methods that use
traditional thermostable RNA ligase, providing triple the output of
competitor kits. Unlike Mth RNA Ligase, which relies on high temperature
(65°C) to minimize oligo circularization and concatemerization events,
Adenylator™ Enzyme performs robustly at 37°C and cannot catalyze
unwanted oligo self-ligation.
Each 10-reaction kit adenylates 3 nmol of DNA or RNA oligos in a
one-step, one-hour, scalable workflow. The resulting 5’-adenylated
oligos are compatible with T4 RNA Ligase 2 truncated or
truncated/K227Q-based reactions for ligation to the 3’-end of RNA.
Adenylator™ adenylated DNA oligos can be used in next-generation
sequencing workflows (e.g., small RNA library prep and Native elongating
transcript sequencing [NET-seq]) and adenylated RNA oligos can be used
as an input into RNA circularization workflows or in a variety of RNA
studies.
Product Performance:
Figure 1. DNA and RNA oligonucleotides are efficiently
adenylated using the Adenylator™ DNA/RNA 5’-Adenylation Kit.
Phosphorylated DNA (Fig 1A) and RNA (Fig 1B) oligonucleotides were
adenylated using Adenylator™ Enzyme and visualized on polyacrylamide
gels (20% acrylamide, 8 M urea, 1X TBE, stained with SYBR® Gold). The
untreated oligos (lane 2) were efficiently adenylated (lane 3) as
evidenced by the band shift.
Figure 2. Adenylator™ Enzyme does not catalyze unwanted
oligo self-ligation. A 23-nt phosphorylated DNA oligo was adenylated
using Adenylator™ Enzyme and Mth RNA Ligase and visualized on a
polyacrylamide gel (20% acrylamide, 8 M urea, 1X TBE, stained with SYBR®
Gold). The Mth RNA Ligase adenylated DNA oligo (T-M) contains oligo
concatemer (red arrows) and circularized oligo (blue
arrow) ligation byproducts. Adenylation using Adenylator™ Enzyme (T-A)
cannot form substrate concatemers or circularize oligo substrates,
resulting in adenylated oligos without byproducts.
Materials Supplied:
Important Store at -20°C in a freezer without a defrost cycle. Do not store at –70°C.
|
Adenylator™ DNA/RNA 5'-Adenylation Kit Contents (10 reactions) |
|
Kit Component |
Reagent Volume |
Adenylator™ Enzyme
in 50% glycerol, 50 mM Tris-HCl, pH 7.5, 0.25 M NaCl, 1 mM
dithiothreitol, 0.1 mM EDTA and 0.1% Triton® X-100 |
20 μl |
|
10X Adenylation Reaction Buffer |
20 μl |
|
10 mM ATP |
20 μl |
|
50 μM Control Phosphorylated Oligo |
12 μl |
|
RNase-Free Water |
500 μl |
Materials Required, but not Supplied
- 5′-monophosphorylated DNA or RNA oligonucleotide (100 μM)
- Isopropanol or ethanol (for salt/alcohol precipitation purification of the adenylated reaction product)
- 3 M NaOAc (pH 5.2), 5 M NaCl, 5 M LiCl or 5 M NH4OAc (for salt/alcohol precipitation purification of the adenylated reaction product)
- 70% Ethanol
- Optional: Colored nucleic acid coprecipitant (e.g., Glycoblue™ Coprecipitant [Thermo Fisher] or Pellet Paint® [Sigma Millipore])
- Optional: Materials for polyacrylamide gel electrophoresis
- Optional: ScriptGuard™ RNase Inhibitor (sold separately) if adenylating RNA oligos
Terms & Conditions: The Product listed is currently offered by CELLSCRIPT™ for research use only under the defined Terms and Conditions.
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