New England Biolabs, M0315L, Terminal Transferase

Terminal transferase catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules. Related Categories DNA Labeling,, DNA Manipulation Applications A-tailing,, PCR Specification Unit Definition One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol dTTP into acid-insoluble material in a total reaction volume of 50ul in 1 hour at 37°C using d(A) 18 as primer. Reaction Conditions 1X Terminal Transferase Reaction Buffer Pack Supplement with 0.25 mM CoCl2 Incubate at 37°C 1X Terminal Transferase Reaction Buffer Pack 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate (pH 7.9 @ 25°C) Storage Buffer 50 mM KPO4 100 mM NaCl 1.43 mM β-ME 50% Glycerol 0.1% Triton® X-100 pH 7.3 @ 25°C Heat Inactivation 75°C for 20 minutes Molecular Weight Theoretical: 58000 daltons 5' - 3' Exonuclease No 3' - 5' Exonuclease No Strand Displacement No Unit Assay Conditions 1X Terminal Transferase Reaction Buffer, 0.72 μM d(A) 18 , 0.2 mM dTTP, and 1 μCi [ 3 H]- dTTP in a 50 μl total reaction volume. FAQ Q: Can Terminal Transferase be heat inactivated? A: Yes, heat to 75°C for 20 minutes. Alternatively, reactions can be stopped by adding 10 μl of 0.2 M EDTA. Q: Can Terminal Transferase label the 5' end of DNA? A: No. Use T4 Polynucleotide Kinase (NEB# M0201) to label the 5' end. Q: Is Terminal Transferase supplied with dNTPs? A: No, the dNTPs must be ordered separately. They can be ordered as a set of 4 individual tubes, Deoxynucleotide Solution Set (NEB# N0446) with a 100mM concentration of each nucleotide. Q: Does Terminal Transferase require Co2+ as a divalent cation? A: Co2+ increases the incorporation of pyrimidines (Maniatis), and makes addition to blunt and 3' recessed ends more efficient (Current Protocols). Q: Can just one dNTP be added to the end of DNA with Terminal Transferase? A: Yes, single nucleotides can be added by incorporation of a chain terminating analog such as a dideoxynucleotide. (ddATP) Q: Does Terminal Transferase have a preference for one type of 3' DNA end? A: Addition of dNTPs to 3' OH protruding ends is more efficient than with 3' OH recessed or blunt ends. Q: What are the kM values for Terminal Transferase? A: * 100 μM for dATP * 100 μM for dTTP * 500 μM for dCTP * 500 μM for dGTP * 1 μM for oligos * 1 mM for homopolymer primer ends Q: Will Terminal Transferase add a non-radioactively labeled dNTP? A: Yes, Terminal Transferase will incorporate biotinylated nucleotides.