New England Biolabs, M1524L, TET2

Related Categories Base Modifying Enzymes,, DNA Methylation Analysis,, DNA Modifying Enzymes & Cloning Technologies Specification Materials Required but not Supplied Monarch PCR & DNA Cleanup Kit (NEB #T1130), or Thermolabile Proteinase K (NEB #P8111) Nuclease-free Water (NEB #B1500) Unit Definition One unit is defined as the amount of enzyme required to modify 1.4 pmol of a 21-mer fluorescently labeled oligonucleotide duplex containing a central methylated CpG moiety in a total reaction volume of 20 μl in 1 hour at 37°C. Reaction Conditions 1X TET2 Reaction Buffer Incubate at 37°C Heat Inactivation No Unit Assay Conditions A fluorescently labeled dsDNA oligonucleotide containing a single 5mC is incubated with 1 unit of TET2 in 1X TET2 Reaction Buffer (containing supplement and Fe2+). 1 unit of TET2 must modify ≥80% of 1.75 pmol of substrate as determined by capillary electrophoresis. FAQ Q: Can TET2 (NEB #M1524) be heat inactivated? A: No, TET2 is not readily inactivated by heat. TET2 may be removed from the reaction using a column for cleanup (such as the Monarch® PCR & DNA Cleanup Kit, (NEB #T1130) or by treatment with Thermolabile Proteinase K (NEB #P8111)). Q: Can the freshly diluted Fe(II) Solution be stored long term? A: No. The freshly diluted Fe(II) solution should be discarded immediately after use. Q: Can the reconstituted 1X TET2 Reaction Buffer Supplement (supplied with NEB #M1524) be stored longer than 4 months? A: No, the reconstituted 1X TET2 Reaction Buffer Supplement should be discarded after 4 months. It is recommended that the date of reconstitution be recorded on the vial. Q: Does TET2 (NEB #M1524) exhibit any sequence preference? A: Yes, TET2 can display some sequence preference, but only at low enzyme concentrations (e.g., highest activity in a CpG context relative to a CHH or CHG context). Sequence preferences can be overcome by using at least 8 U (1 µl of enzyme). Q: How long can the lyophilized TET2 Reaction Buffer Supplement (supplied with NEB #M1524) be stored? A: At -20°C, the lyophilized TET2 Reaction Buffer Supplement remains viable for up to two years. However, once reconstituted in 5X TET2 Reaction Buffer, the TET2 Reaction Buffer Supplement should be discarded after 4 months of storage at -20°C Q: Is it normal if the Fe(II) solution is yellow or a color change is observed? A: Yes, color variation (colorless to yellow) has been observed. Testing has shown no difference in performance based on this variation. Q: Is TET2 (NEB #M1524) active on asymmetrically methylated substrates? A: Yes, the methylation status of the complementary strand does not impact TET2 activity. Q: Is TET2 (NEB #M1524) active on methylated RNA? A: No, TET2 is not active on either single stranded or double stranded RNA. Q: Is TET2 (NEB #M1524) active on methylated single stranded DNA? A: Yes, TET2 is active on both single and double stranded DNA, with a preference for double stranded DNA. Q: Is TET2 (NEB #M1524) inhibited by EDTA? A: Yes, EDTA is inhibitory toward TET2 activity. We recommend keeping EDTA concentrations below 0.01 mM (final) in the TET2 reaction. Q: Is TET2 (NEB #M1524) inhibited by salt? A: Yes, TET2 is inhibited by salt. We recommend keeping salt concentrations below 50 mM (final) in the TET2 reaction. Q: What is the activity of TET2 (NEB #M1524) in other buffers? A: TET2 Reaction Buffer is specifically formulated for optimal TET2 activity. Other buffers are not recommended for use with TET2. Q: What is the reaction temperature range for TET2 (NEB #M1524)? A: TET2 is active at temperatures between 4-60°C, with 37°C being optimal.