New England Biolabs, M0204L, T4 RNA Ligase 1 (ssRNA Ligase)

There is also a high concentration (30,000 units/ml) version of this enzyme available, T4 RNA Ligase 1 (ssRNA Ligase), High Concentration ( Related Categories RNA Ligation Applications RNA Cloning Specification Materials Required but not Supplied Nuclease-free Water (NEB #B1500) RNase Inhibitor, Murine (NEB #M0314) (optional) Unit Definition One unit is defined as the amount of enzyme required to convert 1 nanomole of fluorescently labeled rA16 into a phosphatase-resistant form in 30 minutes at 37°C. Reaction Conditions 1X T4 RNA Ligase Reaction Buffer Supplement with 1 mM ATP Incubate at 25°C 1X T4 RNA Ligase Reaction Buffer 50 mM Tris-HCl 10 mM MgCl2 1 mM DTT (pH 7.5 @ 25°C) Storage Buffer 10 mM Tris-HCl 50 mM KCl 1 mM DTT 0.1 mM EDTA 50% Glycerol pH 7.4 @ 25°C Heat Inactivation 65°C for 15 minutes Unit Assay Conditions 1X T4 RNA Ligase reaction buffer, supplemented with 1 mM ATP, is mixed with the RNA substrate (10µM of fluorescently labeled rA16 ) and varying amounts of enzyme. Incubation is at 37°C for 15 minutes (8). Concentration: 10,000 units/ml FAQ Q: What is the optimal reaction temperature and time for T4 RNA Ligase I? A: In general, increased reaction time and lowered reaction temperature yield more complete ligation reactions. A typical ligation reaction should be carried out at 25°C for up to 2 hours. For longer oligos, 2 hours of incubation at 25°C followed by overnight incubation at 16°C may improve yield. Q: Can T4 RNA Ligase 1 be used to end label single stranded DNA or RNA? A: T4 RNA Ligase 1 can be used for 3' end labeling of ssRNA. Terminal Transferase (#M0315) can be used for 3' end labeling of ssDNA. T4 PNK (#M0201) can be used for 5' end labeling of ssDNA. Q: Will T4 RNA Ligase 1 ligate double stranded DNA? A: No, not significantly. Contrary to early reports T4 RNA Ligase 1 does not improve blunt end ligation significantly when used with T4 DNA Ligase 1. (1). (1) Sugino, A. et al. (1977) J. Biol. Chem. 252, 3987-3994. Q: What is the most common cause of ligation failure when using T4 RNA Ligase 1? A: RNA secondary structure shielding the ends can inhibit ligation. Addition of DMSO to 10% (v/v) can increase ligation in these cases. Q: Why doesn’t the new T4 RNA Ligase reaction buffer contain ATP? A: To allow customers more flexibility in the use of this product we now provide 10 mM ATP and 50% PEG8000 solutions as separate components from the enzyme buffer. The T4 RNA Ligase Reaction Buffer (NEB #B0216) is also compatible with T4 RNA Ligase 2, truncated (NEB #M0242) and T4 RNA Ligase 2, truncated K227Q (NEB #M0351). Q: Will the ligation reaction using T4 RNA Ligase 1 produce circles or duplex products? A: The reaction can produce both. The unit is defined by producing circles using rA-20mer as a substrate. Increasing the concentration, length and adding PEG can increase the number of intermolecular ligations. Blocking one end of the molecule with a dideoxy terminator will prevent the molecule from forming a circle. Q: Can T4 RNA Ligase 1 be used to make single stranded RNA/DNA hybrids? A: Yes. Q: Can T4 RNA Ligase 1 ligate triphosphates? A: No. However, the RNA can be treated first with CIP to remove the triphosphate, and then with a kinase, to add a single phosphate. Fragments containing these 5’ monophosphates can be ligated by T4 RNA ligase 1. Q: Can T4 RNA Ligase 1 be used to add tails to linear duplex DNA? A: Yes, RNA oligos can be ligated to blunt or cohesive end duplex DNA. DNA oligos ligate at a greatly reduced rate (3). (3) Higgins, N. P. et al. (1979) NAR 6, pp. 1013-1024. Q: Can a dideoxy be used to block ligation using T4 RNA Ligase 1? A: Yes (2). (2) Troutt, A. B., et al. (1992) Proc. Natl. Acad. Sci. USA 89. pp. 9823-9825. Q: Can T4 RNA Ligase 1 be used to incorporate unnatural amino acids into proteins? A: Yes, see the reference Noren, C.J. et al. (1989) Science 244, 182-188. Q: Will PEG improve ligation with T4 RNA Ligase 1? A: Yes (4), Quick Ligation Kit buffer (NEB# M2200) can be used with T4 RNA Ligase 1. (4) Harrison, B., and Zimmerman, S. B., (1984) N.A.R. 12, 8235-8251. Q: Will T4 RNA Ligase 1 ligate single stranded DNA? A: Yes. The reaction is slower than RNA/RNA or RNA/DNA. For example, primers can be ligated onto cDNA (2). (2) Troutt, A. B., et al. (1992) Proc. Natl. Acad. Sci. USA 89. pp. 9823-9825. Q: Is PEG 8000 available for purchase? A: PEG 8000 is included as a component of the T4 RNA Ligase Reaction Buffer (NEB #B0216). Q: What is the salt tolerance of this RNA ligase? A: NEB # RNA Ligase Salt Tolerance M0204 T4 RNA Ligase 1 (ssRNA Ligase) ≤ 50 mM M0239 T4 RNA Ligase 2 (dsRNA Ligase) ≤ 50 mM M0242 T4 RNA Ligase 2, truncated ≤ 100 mM M0351 T4 RNA Ligase 2, trunc. K277Q ≤ 100 mM M0373 T4 RNA Ligase 2, trunc. KQ ≤ 100 mM M0375 SplintR Ligase ≤ 50 mM E2610 5' Adenylation Kit (Mth Ligase) ≤ 300 mM M0319 Thermostable 5' App DNA/RNA Ligase ≤ 50 mM