New England Biolabs, M0373S, T4 RNA Ligase 2, truncated KQ

T4 RNA Ligase 2, truncated KQ ligates the pre-adenylated 5´ end of DNA or RNA to the 3´ end of RNA. Related Categories RNA Ligation,, RT-PCR,, cDNA Synthesis & Reverse Transcriptases Applications RNA Cloning,, RT-PCR & cDNA Synthesis,, PCR Specification Unit Definition 200 units is defined as the amount of enzyme required to give 80% ligation of a 31-mer RNA to the pre-adenylated end of a 17-mer DNA [Universal miRNA Cloning Linker ( NEB #S1315 )] in a total reaction volume of 20 μl in 1 hour at 25°C. 5´-FAM- rArGrUrCrGrUrArGrCrCrUrUrUrArUrCrCrGrArGrArUrUr CrArGrCrArArUrA-3´ 5´-rAppCTGTAGGCACCATCAAT–NH2-3´ Reaction Conditions 1X T4 RNA Ligase Reaction Buffer 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 ml Tris-HCl 100 mM NaCl 0.1 mM EDTA 1 mM DTT 50% Glycerol pH 7.5 @ 25°C Heat Inactivation 65°C for 20 minutes Molecular Weight Theoretical: 71378.96 daltons Specific Activity 200,000 units/mg Unit Assay Conditions 1X T4 RNA Ligase Reaction Buffer supplemented to 10% (w/v) PEG MW 8000, 20 pmol of 5´-FAM labeled RNA, and 40 pmol preadenylated DNA linker. After incubation at 25°C for 1 hour, the ligated product is detected on a 15% denaturing polyacrylamide gel. FAQ Q: Is T4 RNA Ligase 2, truncated KQ different than T4 RNA Ligase 2, truncated K227Q? A: Yes. T4 RNA Ligase 2, truncated KQ combines 2 active site mutations - R55K and K227Q, that improve the performance of this enzyme as compared to T4 RNA Ligase 2, truncated and T4 RNA Ligase 2, truncated K227Q. Ligation with T4 RNA Ligase 2, truncated KQ reduces levels of side ligation products to levels comparable to T4 RNA Ligase 2, truncated K227Q, while accumulating desired ligation products at rates similar to T4 RNA Ligase 2, truncated. Q: Are there differences in ligation efficiency for T4 RNA Ligase 2, truncated KQ, T4 RNA Ligase 2, truncated 227Q, and T4 RNA Ligase 2, truncated KQ? A: In NEB tests, ligations using T4 RNA Ligase 2, truncated T4 RNA Ligase 2, truncated K227Q, and T4 RNA Ligase 2, truncated KQ on identical substrates show that ligation efficiency reaches the same endpoints. While, T4 Ligase 2, truncated KQ and T4 RNA Ligase 2, truncated require similar incubation times, T4 RNA Ligase 2, truncated K227Q may require longer incubation times to reach the reaction endpoint. Q: What can T4 RNA Ligase 2, truncated KQ ligate? A: T4 RNA Ligase 2, truncated K227Q can perform the same ligations as T4 RNA Ligase 2, truncated (NEB #M0242). Q: Does PEG stimulate ligation efficiency? A: PEG 8000 greatly stimulates the ligation activity of T4 Rnl2tr K227Q. Maximum stimulation was observed at 15% (w/v) PEG 8000. PEG 4000 is also stimulatory with a similar concentration effect to PEG 8000. Q: How can I increase ligation efficiency? A: In general, increased reaction time, lowered reaction temperature, and molecular crowding all yield more complete ligation reactions. Reaction times can be increased as long as 24 hours or more. For ligations longer than 2 hours, we routinely incubate below 16°C. Some scientists report ligation on ice (0°C) for 24 hours. We recommend the use of PEG 4000 or 8000 up to 15% (w/v). Increasing PEG concentrations further did not further increase ligation efficiency. Q: Can T4 RNA Ligase 2, truncated KQ be used in other NEBuffers? A: Optimal activity is seen in its unique buffer, however, the enzyme is active in NEBuffer 1 and 2. Q: The molecular weight of T4 RNA Ligase 2, truncated KQ is 71.6 kDa. Is it a fusion? A: Yes. In our tests, the MBP fusion, and untagged enzymes perform identically. 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