Universal CG cloning of polymerase chain reaction products

Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 3′ A to a plasmid vector containing a 3′ T. In this article, we show that the analogous “C...

Full description

Saved in:
Bibliographic Details
Published in:Analytical biochemistry 2015-02, Vol.471, p.80-82
Main Authors: Stevenson, Julian, Brown, Andrew J.
Format: Article
Language:English
Subjects:
Cloning
Cloning, Molecular - methods
DNA - genetics
PCR
Plasmid vectors
Polymerase Chain Reaction
Time Factors
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Single-insert cloning of DNA fragments without restriction enzymes has traditionally been achieved using TA cloning, with annealing of a polymerase chain reaction (PCR) fragment containing a single overhanging 3′ A to a plasmid vector containing a 3′ T. In this article, we show that the analogous “CG cloning” is faster and far more efficient, using AhdI to generate a C-vector. For an afternoon ligation, CG cloning achieved double the cloning efficiency and more than 4-fold the number of transformants compared with TA cloning. However, blunt-end ligation was markedly more efficient than both. CG cloning could prove to be extremely useful for single-copy high-throughput cloning.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2014.10.018