Using a modified TA cloning method to create entry clones

doi:10.1016/j.ab.2006.08.015

Analytical Biochemistry

Volume 358, Issue 1, 1 November 2006, Pages 120-125

https://doi.org/10.1016/j.ab.2006.08.015 Get rights and content

Abstract

We describe a noncommercial alternative method to create entry clones compatible with all kinds of destination vectors based on an improved TA cloning approach. To generate Gateway T vectors, we first constructed gentamicin- and chloramphenicol-resistant entry vectors designated pGWG and pGWC, respectively. Each entry vector contains an AhdI cassette flanked by attL sites, with each AhdI cassette containing two AhdI restriction enzyme sites spaced by the ccdB killer gene, which is lethal to most Escherichia coli strains. Gateway T vectors can be prepared by simple digestion of these entry vectors with the AhdI enzyme or its isoschizomers. The use of the ccdB gene as a negative selection marker is an important improvement over conventional TA cloning in that it eliminates the necessity of blue/white color screening based on α-complementation. Another important improvement that we have implemented is to retail the T vectors using Taq polymerase and dTTP so as to improve the cloning efficiency. Together, these improvements allow TA cloning to realize its full potential. Using Gateway T vectors prepared by this improved method, entry clones for PCR products or restriction enzyme fragments can be created simply, efficiently, and inexpensively while at the same time introducing greater compatibility.

Section snippets

Vector construction

The pGWG vector was derived sequentially from the pGWGP, pGL12, pGEN, pGL12P, and pENTR1A intermediate vectors (Invitrogen). The sequences of pGWG and pGWC are provided in the supplementary data. All of the ccdB-containing vectors were maintained and propagated in Escherichia coli strain DB3.1 (Invitrogen), and all PCR products were amplified with proofreading Pfu DNA polymerase and verified by sequencing when necessary. All primer pairs described below are shown in Table 1.

To construct pGL12P,

Construction of gentamicin- and chloramphenicol-resistant entry vectors using ccdB killer gene as a negative selection marker

To generate Gateway T vectors with enhanced compatibility, Gen- and chloramphenicol (Chl)-resistant entry vectors, pGWG and pGWC, were constructed (Fig. 1). These entry vectors are compatible with all kinds of destination vectors, including those with Amp-, Kan-, or Spe-resistant genes as screening markers. pGWC has the best compatibility because all of the expression clones derived from destination vectors carry no Chl-resistant genes. Each entry vector contains an attL site-flanked AhdI

Discussion

The experiments described here demonstrate that entry clones for PCR products or restriction enzyme fragments can be created in a simple, efficient, and inexpensive method along with better compatibility using an improved TA cloning method together with Gateway T vectors. In this article, we described two important improvements for TA cloning: (i) the use of the negative selection marker gene ccdB to separate the two AhdI restriction enzyme sites and (ii) the retailing of the Gateway T vectors

Acknowledgments

The work was supported by the National Basic Research Program of China (2006CB100100) and the National Natural Science Foundation of China (30300025).

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Analytical Biochemistry

Abstract

Section snippets

Vector construction

Construction of gentamicin- and chloramphenicol-resistant entry vectors using ccdB killer gene as a negative selection marker

Discussion

Acknowledgments

References (14)

GATEWAY vectors for Agrobacterium-mediated plant transformation

Trends Plant Sci.

Construction of a set of Gateway-based destination vectors for high-throughput cloning and expression screening in Escherichia coli

Anal. Biochem.

GATEWAY vectors for Plasmodium falciparum transfection

Trends Parasitol.

New donor vector for generation of histidine-tagged fusion proteins using the Gateway Cloning System

Plasmid

New ccdB positive-selection cloning vectors with kanamycin or chloramphenicol selectable markers

Gene

DNA cloning using in vitro site-specific recombination

Genome Res.

A gateway cloning vector set for high-throughput functional analysis of genes in planta

Plant Physiol.

Cited by (34)

Expressing a Short Tandem Target Mimic (STTM) of miR164b/e-3p enhances poplar leaf serration by co-regulating the miR164–NAC module

Expression pattern of GmLAX genes under different stresses in soybean drought sensitive cultivar and tolerant cultivar

GmDRR1, a dirigent protein resistant to Phytophthora sojae in Glycine max (L.) Merr.

A callus transformation system for gene functional studies in soybean

The Miscanthus NAC transcription factor MlNAC9 enhances abiotic stress tolerance in transgenic Arabidopsis

Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3