Blunting of DNA ends and Ligation

Blunting of DNA ends

DNA fragment (0.5-1.0 μg) 1-16 μl
T4 DNA polimerase   1 μl
10X buffer   2 μl
ddH2O up to 20 μl
  1. Incubate 30 min at 37°C
  2. Add 80 μl H2O, 100 μl phenol/chloroform pH 8 (3:1), vortex and spin for 1 min
  3. To the upper phase add 100 μl chloroform, vortex and spin again
  4. To the upper phase add 1 μl glycogen, 10 μl 3 M NaAc pH 5 and 220 μl EtOH. Keep 15 min at –70°C
  5. Centrifuge for 10 min at max. speed. Wash the pellet with 70% EtOH
  6. Resuspend DNA in 20 μl H2O

Ligation (equal amounts of vector and fragment ends):

50-500 ng Blunted/Kinased DNA fragment   1-22 μl
50-100 ng dephosphorylated vector   1-2 μl
10 X ligation buffer   3 μl
50% PEG 4000 (10X)   3 μl
T4 DNA ligase (~4U)   1 μl
dd H2O   up to 30 μl
  1. Incubate 1 h at 22 °C
  2. Inactivate by heating 10 min at 65 °C
  3. Extract with chloroform
  4. EtOH precipitate
  5. Resuspend in a few μl of H2O and transform

To calculate the amount of ends (in pmol):
pmol ends = [2 × DNA mass (in ng) × 103]/[bp DN A × 649 daltons]

Ex: 50 ng pU C19 → [2 × 50ng × 1000]/ [2686bp × 649]  = 0.057 pmol of ends