The enzyme deoxynucleotidyl terminal transferase (TdT) catalyses the addition of dNTPs onto the 3’ ends of double stranded DNA molecules. In an experiment to determine the rate at which the TdT added dNTPs to the 3’ end of DNA, the following experiment was set up:
Linear-bacteriophage lambda DNA (48.5kb) at a concentration of 55ug ml^-1 was cut with the restriction enzyme Alu I. (Lambda DNA is cut 143 times with Alu I).
The following reaction was set up:
17ul H2O (ul = microlitres)
6ul 5x tailing buffer
2ul Alu I cut lambda DNA
2ul 1mM dCTP
2ul 32p DTP
1ul (15U) of TdT
The reaction mix was incubated at 37C and after 5min, 2ul aliquots were removed from the reaction mix to determine the amount of radioactivity incorporated. One aliquot was counted directly to determine the total amount of radioactivity in the sample. Another aliquot was TCA precipitated to determine the amount of radioactivity incorporated into the DNA. The results were as follows:
Counts before TCA precipitation = 374248 cpm
Counts after TCA precipitation = 4905 cpm
If the reaction was to be repeated, how many minutes would you allow the reaction to continue if you only wanted the addition of 20 bases of dCTP to each 3’ end of the DNA fragments.
5x tail buffer : 500mM potassium cacodylate pH 7.2, 10mM cobalt chloride, 1mM DTT
32PdCTP;5uCi ul-1 (3000 Ci mMol-1)
(molecular weight of 1.6kb of dsDNA = 1×106)