The USER-friendly DNA engineering method allows combining multiple PCR fragment assembly, nucleotide sequence alteration and directional cloning. In this approach, target DNA molecules and cloning vector are generated by PCR with 6-10 bases of homology between the neighboring fragments. PCR primers contain a single deoxyuracil residue (dU) flanking the 3’ end of the homology region, and can be designed to accommodate nucleotide substitutions, insertions and/or deletions. The primers are then used to amplify the vector and target DNA in discrete overlapping fragments that incorporate a dU at each end. The subsequent treatment of PCR fragments with USER Enzyme creates a single nucleotide gap at each location of dU resulting in PCR fragments flanked with single-stranded extensions that allow seamless and directional assembly of customized DNA molecules into a linearized vector. In this way, multi-fragment assemblies, as well as various mutagenic changes, can all be accomplished in a single-format experiment.
- Bitinaite J. et al. (2007) Nucl.Acids Res.35,1992-2002.
- Bitinaite, J. and Nichols, N.M. (2009) Curr Protoc Mol Biol., Chapter 3, Unit 3.21.1-3.21.16
- Vaisvila, R. and Bitinaite, J. (2013) Methods Mol.Biol. 978, 165-171.
USER® Cloning includes these areas of focus:
Protocols for USER® Cloning
- 14 Minute Transformation Protocol (C2987H/C2987I)
- 5 Minute Transformation Protocol (C2987H/C2987I)
- 5 Minute Transformation Protocol using NEB® 10-beta Competent E. coli (C3019)
- Cloning with USER Enzyme
- High Efficiency Transformation Protocol (C2987H/C2987I)
- High Efficiency Transformation Protocol for 384-well format (C2987R)
- High Efficiency Transformation Protocol for 96-tube format (C2987U)
- High Efficiency Transformation Protocol for 96-well format (C2987P)
- High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (High Efficiency) (C3019)
Molecular Cloning Technical Guide
Download the latest Molecular Cloning Technical Guide for help with product selection, protocols, tips for optimization and trouble-shooting.
Reagents & Tools for Molecular Cloning brochure
Learn about recommended products for cloning in our Reagents and Tools for Molecular Cloning Brochure.
- Synthetic Biology/DNA Assembly Selection Chart
Other Tools & Resources
- Lund AM, Kildegaard HF, Petersen MB, Rank J, Hansen BG, Andersen MR, Mortensen UH 2014. A versatile system for USER cloning-based assembly of expression vectors for mammalian cell engineering PLoS One. 9(5), PubMedID: 24879460, DOI: 10.1371/journal.pone.0096693
- Wang S, Li W, Wang S, Hu B 2014. Rapid and Efficient Assembly of Transcription Activator-Like Effector Genes by USER Cloning J Genet Genomics. 41(6), PubMedID: 24976123, DOI: 10.1016/j.jgg.2014.05.002
- Villiers BR, Stein V, Hollfelder F 2010. USER friendly DNA recombination (USERec): a simple and flexible near homology-independent method for gene library construction Protein Eng Des Sel. 23(1), PubMedID: 19897542, DOI: 10.1093/protein/gzp063
Publications related to USER® Cloning
While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.
For more information about commercial rights, please contact NEB's Global Business Development team at firstname.lastname@example.org.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.