Protocol abstract
Nature Protocols 2, - 2198 - 2202 (2007)
Published online: 6 September 2007 | doi:10.1038/nprot.2007.325
Subject Category: Nucleic acid based molecular biology
Enzymatic ligation assisted by nucleases: simultaneous ligation and digestion promote the ordered assembly of DNA
Gregory J Cost1
Abstract
This protocol describes a method for the one-tube preparative-scale assembly of a specific DNA molecule, the enzymatic ligation assisted by nucleases (ELAN) technique. DNA fragments in ligation reactions are capable of combining to produce numerous products. The ELAN method uses judicious choice of restriction enzyme sites coupled with simultaneous digestion and ligation reactions to create just one product, by converting off-pathway products back into substrate. The experimental parameters critical for a successful ELAN reaction are discussed, and the ordered, one-tube assembly of four DNA fragments in the presence of eight restriction enzymes is demonstrated. This technique will be useful to those performing gene construction, DNA computing, biophysics and even standard molecular cloning. Starting with reactant fragments, the protocol takes 4–16 h to produce nanogram to microgram yields, depending on the complexity of the reaction.
- Department of Molecular and Cell Biology, University of California, Berkeley, California, USA. Current address: Sangamo BioSciences, 501 Canal Boulevard, Richmond, California 94804, USA.
nature-products
A-Z product listing
- 10
TAE(Sigma) - Agarose(Pharmacia)
- ATP(Pharmacia)
- BSA(New England Biolabs)
- DNA fragment purification spin columns(Qiagen)
- DNA molecular weight ladder(New England Biolabs)
