Blunt End Cloning of PCR Products PCR产物平端克隆

Hancock Laboratory Methods. Department of Microbiology and Immunology,

University of British Columbia, British Columbia, Canada

REFERENCE: GATA 1993; 10(5):113-115.

1.End repair: Add 5-10 units of T4 DNA pol and incubate at 37ºC 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 14,000 x g for 20 minutes.

3.Wash with 70 % ethanol, dry and resuspend in 20μl T4 polynucleotide kinase, containing 20 pmol ATP and 5-10 units T4 polynucleotide kinase. Incubate at 37ºC 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 50μl reaction mixture containing 1 unit of T4 ligase. Do ligation overnight using a temperature gradient of 25― 2ºC.