New England Biolabs, E2610L, 5´ DNA Adenylation Kit

Related Categories RNA Ligation FAQ Q: Can this 5' DNA adenylation kit be used for adenylating ssRNA oligos? A: Yes, this kit can adenylate the 5' end of ssRNA oligos. Q: What is the molecular weight of Mth RNA Ligase? Is it a fusion protein? A: Mth RNA Ligase is a His10 fusion protein with a molecular weight of 46 kDa. Q: What ratio of enzyme to oligonucleotide do you recommend A: We recommend a one to one molar ratio of enzyme to oligonucleotide for adenylation of the most difficult substrates. For some oligonucleotides, 2-4 fold less enzyme is sufficient. Q: Can Mth RNA Ligase be used in other NEBuffers? A: No. Optimal labeling efficiency for this enzyme is achieved only in the temperature-independent 5´ DNA Adenylation Reaction Buffer supplied with the product. pH 6.0 is critical. Q: Can Mth RNA Ligase be used for ligation? A: No. Reaction conditions are optimized for 5’-DNA adenylation. Q: Does PEG stimulate adenylation? A: No. PEG does not stimulate adenylation. Q: What concentration of ATP should be used in the adenylation reaction? A: A range from 0.05 to 0.5 mM ATP can be used. 0.1 mM ATP is optimal for the recommended reaction condition. Q: Will the enzyme work at low temperature (e.g., 37°C)? A: Mth RNA ligase requires elevated temperature for activation, with optimum activity at 65°C. Lowering the temperature will require an extension of the reaction time, which should be determined empirically. For purification of ssDNA, be sure to follow recommendations detailed in T1030 FAQ#8. Q: How do I determine the extent of adenylation? A: An adenylated oligonucleotide runs about 1 base slower on a denaturing 15 or 20% polyacrylamide gel. If the 5´-phosphate is labeled, it also becomes phosphatase resistant For oligonucleotides >18 bases, the Monarch PCR & DNA Cleanup Kit (T1030) is the recommended product for purifying adenylated oligos after the reaction. The protocol is available through the following link: Monarch® PCR & DNA Cleanup Kit (5 μg) Q: Do I need to clean up the adenylation reaction before I use the oligo for ligation? A: We recommend heat or proteinase K treatment to inactivate enzyme. Excess ATP should be removed from the reaction, either by precipitation or column chromatography, to prevent interference with the subsequent ligation step. Q: What is the composition of the 5´ DNA Adenylation Reaction Buffer at 1X concentration? A: The 5´ DNA Adenylation Reaction Buffer at 1X concentration is: 50 mM Sodium Acetate (pH 6.0 @ 25°C) 10 mM MgCl2 5 mM DTT 0.1 mM EDTA 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 Q: How can I scale up my adenylation reaction? A: For larger-scale applications, you can increase the reaction volume and use our High Concentration Mth RNA Ligase, available exclusively through the Custom Solutions channel. This allows the adenylation reaction to be scaled up to 30 pmol of oligonucleotide and 30 pmol of enzyme per µl without loss of efficiency. After the reaction, the oligonucleotide can be purified by phenol extraction and alcohol precipitation or column chromatography to effectively remove protein and ATP.