DNA ligation_生物行

Wednesday, October 08, 2003

Description
DNA ligations are performed by incubating DNA fragments with appropriately linearized cloning vector in the presence of buffer, rATP, and T4 DNA ligase (1,2). For random shotgun cloning, sonicated or nebulized fragments are ligated to either SmaI linearized, dephosphorylated double-stranded M13 replicative form or pUC vector by incubation at 4degC overnight. A practical range of concentrations is determined based on the amount of initial DNA, and several different ligations, each with an amount of insert DNA within that range, are used to determine the appropriate insert to vector ratio for the ligation reaction. In addition, several control ligations are performed to test the efficiency of the blunt-ending process, the ligation reaction, and the quality of the vector (1,2). These usually included parallel ligations in the absence of insert DNA to determine the background clones arising from self-ligation of inefficiently phosphatased vector. Parallel ligations also are performed with a known blunt-ended insert or insert library, typically an AluI digest of a cosmid, to insure that the blunt-ended ligation reaction would yield sufficient insert containing clones, independent of the repair process.

Primary Author
Bruce A. Roe ( broe@ou.edu )

Affiliation
University of Oklahoma , United States

Co-Author(s)
Judy S. Crabtree
Akbar S. Khan

Procedure
1. Combine the following reagents in a microcentrifuge tube, and incubate overnight at 4degC:

DNA fragments: 100-1000 ng
cloning vector: 2 ul (10 ng/ul)
10X ligation buffer: 1 ul
T4 DNA ligase (NEB 202L): 1 ul (400 U/ul)
sterile ddH2O: q.s.
Total: 10 ul

The cloning vector typically is SmaI-linearized, CIAP-dephosphorylated pUC vector (Pharmacia 27-4860-01) as several years ago we switched from M13 to pUC-based shotgun cloning. The advantage of obtaining two sequence reads off one isolated shotgun sub-clone seems to outweigh the disadvantage of a few bases less in double-stranded vs single-stranded read lengths. In some instances, including 5% PEG in the ligation reactions also seems to slightly improve the ligation efficiency.

2. Include control ligation reactions with no insert DNA and with a known blunt-ended insert (such as AluI digested cosmid).

Recipes

Supplies
T4 DNA ligase (NEB 202L)
pUC vector (Pharmacia 27-4860-01)

Tips

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DNA ligation