Blunt end cloning of PCR products

GATA 1993; 10(5):113-115

  1. End repair: Add 5-10 units of T4 DNApol and incubate at 37C for 5 minutes.
  2. Make solution to 2.5 M NH4OAc and add 1 volume of 95% ethanol; let sit for 5 minutes at RT and spin at 14K x g for 20 minutes.
  3. Wash with 70 % ethanol, dry and resuspend in 20 ul T4 PNK, 20 pmol ATP and 5-10 units T4 PNK. Incubate at 37 C for 1 hour.
  4. Ligation reaction: Blunt end ligation is carried out in the presence of 5% PEG 8000. A microgram of Bluescript SK+ is blunt ended (cut with EcoRV) and ligated with 10-100 ng of the purified PCR product in a 50 ul reaction mixture containing 1 unit of T4 ligase. Do ligation overnight using a temperature gradient of 25-2C.