For telomere elongation, 2 μL of telomerase extract with various concentrations ranging from 101 to 106 cells was incubated with 1 μL TS primer (100pmol) in 10 μL reaction buffer (50mM Tris-HCl, 150mM NaCl, 10mM, KCl, 1mM MgCl2, pH 7.4) at 37 °C for 1 h.
I also need dNTPs for this reaction but I am confused about the volume of dNTPs needed? ( dNTPs 1mM)
Hi Le Uyen,
If I'm getting your idea, you want to choose an ideal dNTPs concentration. First you would try a physiological concentration. This values are according to reported evidences =Traut, T. W. Physiological concentrations of purines and pyrimidines. Mol. Cell. Biochem. 140, 1–22 (1994)= : 24 μM dATP, 29 μM dCTP, 37 μM dTTP, 5.2 μM dGTP, or if you would like to see a robust activity you would increase the above concentration up to 200 uM each one (dATP, dGTP, dTTP, dCTP). For a 20 ul reaction you will need to add 1 ul of dNTPs at 1 mM ( you can dilute the dNTPs from 1 to 0.5 mM dNTPs and in that way use 2 ul (10 % volume of your final reaction)
Regards
Noe
Article Assays for Detection of Telomerase Activity
Article One-step affinity purification protocol for human telomerase
Article Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs
Perhaps those to start with. Good luck.
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