Terminal Deoxyribonucleotidyl Transferase 103- Ethanol-precipitate the remaining DNA in the presence of potassium
acetate.
3.2.3.2. TAILING LINEARIZED VECTOR DNA WITH OLIGO(DCS) - Prepare a reaction mixture with:
Linearized vector DNA (dried pellet)
5X C-tailing buffer
0.1 mM dCTPczS
[(z-35S]dCTP (1300 Ci/mmol, 13 gCi/gL)
Distilled water
Terminal transferase (17 U/laL)
10 pmol 3' ends
20
30
2vL
38
10- Spot a 2-gL aliquot onto a DE81 filter disk, dry under a fan, and count
the total radioactivity in a toluene-based scintillant. - Incubate at 20°C, take 2-1aL aliquots every 15 min, and determine as
soon as possible the incorporated amount of dCS per 3'-OH terminus
by using the DE81 filter technique described in Section 3.2.2. When
about 20 dCS residues are incorporated per primer, stop the reaction by
heating to 65°C for 10 min. Chill on ice. - Ethanol-precipitate DNA in the presence of potassium acetate.
- Take an aliquot, cleave with a restriction endonuclease that cuts close
to the extension, and control the tail lengths by polyacrylamide gel elec-
trophoresis. - If necessary, repeat the procedure by incubating for a shorter or
longer time.
3.2.3.3. ANNEALING OF THE TAILED DNA COMPONENTS
AND TRANSFORMATION OF COMPETENT E. COLI CELLS
- Dissolve the (dGS)-tailed insert DNA and the (dCS)-tailed vector DNA
in l0 laL (each) of l0 mM Tris-HC1, pH 7.2, 1 mM EDTA, and 100 mM
NaC1. - Mix aliquots of the two solutions in an approximate molar ratio of 1:1
(3' ends) for each component. - Incubate at 65°C for l0 min in an incubator block.
- Turn off the incubator block, and let slowly cool down to room tem-
perature. - Take an aliquot with about 1 fmol annealed construct for the transfor-
mation of 100 pL competent E. coli cells.
The same protocol is applicable for tailing with unmodified
deoxynucleoside triphosphates. Transformation efficiencies are about
threefold higher with phosphorothioate-tailed constructs than those
obtained with normal dG/dC-tailed constructs.