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Terminal Transferase > FAQ |  | Terminal Transferase FAQ
See the Polymerases FAQ also.
Q1: Can Terminal Transferase be heat inactivated?
Q2: Can Terminal Transferase label the 5' end of DNA?
Q3: Is Terminal Transferase supplied with dNTPs?
Q4: Does Terminal Transferase require Co2+ as a divalent cation?
Q5: What is the rate of addition of dNTPs to the 3´ ends of DNA using Terminal Transferase?
Q6: Can Terminal Transferase work on RNA?
Q7: Can just one dNTP be added to the end of DNA with Terminal Transferase?
Q8: Does Terminal Transferase have a preference for one type of 3' DNA end?
Q9: Will Terminal Transferase add a non-radioactively labeled dNTP?
Q10: What are the kM values for Terminal Transferase?
Q1: Can Terminal Transferase be heat inactivated?
A1: Yes, heat to 70°C for 10 minutes. Alternatively, reactions can be stopped by adding 10 μl of 0.2 M EDTA.
Q2: Can Terminal Transferase label the 5' end of DNA?
A2: No. Use T4 Polynucleotide Kinase (NEB# M0201) to label the 5' end.
Q3: Is Terminal Transferase supplied with dNTPs?
A3: 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.
Q4: Does Terminal Transferase require Co2+ as a divalent cation?
A4: Co2+ increases the incorporation of pyrimidines (Maniatis), and makes addition to blunt and 3' recessed ends more efficient (Current Protocols).
Q5: What is the rate of addition of dNTPs to the 3´ ends of DNA using Terminal Transferase?
A5: The rate of addition of dNTPs and thus the length of the tail is a function of the ratio of 3´ DNA ends: dNTP concentration, and also which dNTP is used. The following values are approximate and are given for a 15 minute incubation at 37°C in the recommended buffer.
1:01 pMol3´ends:μMdNTP yields tail lenghts of dA: 1-5, dC: 1-3, dG: 1-3, dT: 1-5
1:1 pMol3´ends:μMdNTP yields tail lenghts of dA: 10-20, dC: 10-20, dG: 5-10, dT: 10-20
1:5 pMol3´ends:μMdNTP yields tail lenghts of dA: 100-300, dC: 50-200, dG: 10-25, dT: 200-300
1:10 pMol3´ends:μMdNTP yields tail lenghts of dA: 300-500, dC: 100-150, dG: 15-25, dT: 250-350
Q6: Can Terminal Transferase work on RNA?
A6: In the presence of Co2+, Terminal Transferase will add rNTPs to a limited extent (1).
(1) Deng, G.R. and Wu, R. 1983 Meth. Enzymol. 100:96-116.
Q7: Can just one dNTP be added to the end of DNA with Terminal Transferase?
A7: Yes, single nucleotides can be added by incorporation of a chain terminating analog such as a dideoxynucleotide. (ddATP)
Q8: Does Terminal Transferase have a preference for one type of 3' DNA end?
A8: Addition of dNTPs to 3' OH protruding ends is more efficient than with 3' OH recessed or blunt ends.
Q9: Will Terminal Transferase add a non-radioactively labeled dNTP?
A9: Yes, Terminal Transferase will incorporate biotinylated nucleotides.
Q10: What are the kM values for Terminal Transferase?
A10: * 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
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