102 Grosse and Manns- After measuring the total radioactivity, place the filter disk on a suction
device. Rinse several times with ethanol (to remove toluene), and then
dry under a fan. - Keep the dried filter for 5 min in a 0.5M solution of Na2HPO 4. Then
place it on a suction device, and rinse five times with 1 mL (each) of
0.5M Na2HPO 4, two times with 1 mL water, and two times with 1 mL
of ethanol. Then dry under a fan, and count the dCMPS incorporation
in a toluene-based scintillant. - Calculate the amount of dCS-tails added to (dC)10 from the amount of
filter-retained radioactivity:
(cpm binding to DE8 l/Total cpm) ×
(500 ~M dCMPS/10 ]aM 3'-OH) = dCMPS additions/primer (2) - Ethanol-precipitate the remaining DNA in the presence of potassium
acetate. - Control the tail lengths by denaturing polyacrylamide gel electrophoresis.
3.2.3. Tailing of Restriction Fragments with dNTPaS
3.2.3.1. TAILING DOUBLE-STRANDED DNA WITH OLIGO(DGS)
..
3..Prepare a reaction mixture containing:
Double-stranded insert DNA
(dried pellet)
5X G-tailing buffer 5 laL
[~-35S]dGTP (1300 Ci/mmol, 13 ~tCi//aL) 2 pL
100 gM dGTPcxS 6 laL
Distilled water 10 I.tL
Terminal transferase (17 U/gL) 2 ~L
Incubate at 37°C for 30 min.About 2.5 pmol 3' endsSpot a 2-pL aliquot onto a DE81-filter disk, and proceed as described
in Section 3.2.2.
Calculate the amount of dG-tails added to the double-stranded DNA
from the amount of radioactivity bound to the DE81 filter:
(cpm binding to DE81/Total cpm) × (24 ~'/dGMPS/20)
= 3'-OH termini of insert DNA (pM) (3)
If the expected number of 20 for dGMPS added to each 3' terminus is
not achieved or (likewise) the calculated number of 3'-OH termini of
insert DNA is too low, the tailing procedure should be repeated after
ethanol precipitation of the DNA. Because dG- and dGS-containing
tails cannot exceed 20 residues, there is no danger of generating tails
that are too long.