TOPO™ TA Cloning™ Kit for Sequencing, with One Shot™ TOP10 Chemically Competent E. coli

The TOPO™ TA Cloning™ Kits for Sequencing provide a highly efficient, 5-minute, one-step cloning strategy ('TOPO™ Cloning') for the direct insertion of Taq polymerase–amplified PCR products into a plasmid vector for sequencing. Each kit uses the pCR™4-TOPO™ TA vector with specially designed sequencing primer sites that return more insert sequence and less vector sequence from each reaction, and are available with a variety of competent cells, or no competent cells, depending on your needs and budget. These kits include everything necessary to clone and select recombinant vectors containing your PCR fragment of choice. Features of TOPO™ TA Cloning™ Kits for Sequencing:

• Get more sequence—allows for more insert sequence and less vector seuquence when using standard sequencing primers
• Fast and easy—go from PCR to clones in just 3 steps and in as little as 5 minutes hands-on time
• Efficient—obtain up to 95% clones with correct insert
• Proven—reliable performance for over a decade with over 4,000 citations

TOPO™ TA Cloning™ Kits for Sequencing—overview

Vector: pCR™4-TOPO™ TA vector—optimized cloning vector for improved sequencing results

Cloning method: TOPO™ TA Cloning™—Topoisomerase I–based, 5-minute ligation of PCR products with 3´A overhangs (Taq-amplified) to the vector

Competent cells: Various options—choose from kits with general, high-efficiency, bacteriophage T1-resistant, or fast-growing competent cells, or use your own

pCR™4-TOPO™ TA vector—optimized for sequencing
We have removed much of the multiple cloning site from the pCR™4-TOPO™ TA vector to shorten the distance between sequencing primer sites and the insert site to as little as 33 bp. This means sequencing reactions give less vector sequence and more insert sequence. The pCR™4-TOPO™ TA vector has sites for 4 common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.

pCR™4-TOPO™ TA clone selection and manipulation
The pCR™4-TOPO™ TA vector contains both ampicillin and kanamycin resistance markers and a LacZα-ccdB gene fusion for positive selection and blue/white screening. The vector’s minimized multiple cloning site still includes flanking EcoRI sites for simplified excision of cloned PCR products and a unique Sse8387I site for generation of nested deletions prior to sequencing. T7 and T3 promoters are also present for in vitro transcription.

Simplified TOPO™-based cloning
Using TOPO™ cloning technology, there is no need for PCR primers containing specific sequences, post-PCR procedures, vector preparation, or other time-intensive DNA manipulation steps. Just add your PCR reaction straight to the provided topoisomerase-charged vector, incubate 5 minutes, and transform with the providedE. coli competent cells.

Efficient cloning
With up to 95% of clones carrying the desired insert, you can screen less clones and save time and money. The pCR™4-TOPO™ TA vector used in this kit comes with 3´T overhangs for efficient ligation of Taq-amplified PCR products, which contain 3´A overhangs.

The standard in cloning
When it comes to cloning, TOPO™ cloning technology has been a reliable partner for thousands of scientists for over ten years. Fast, simple-to-use, and efficient, TOPO™ cloning has been applied to many different vectors for a wide array of applications.

TOPO™ TA Cloning™ Kits for Sequencing—kit options
The TOPO™ TA Cloning™ Kit for Sequencing can be purchased with a variety of competent cells that deliver different advantages depending upon your needs:

• General cloning: TOP10 (Cat. No. K4575-J10, K4575-01, K4575-40)
• High-efficiency cloning: TOP10 Electrocomp™ Cells (Cat. No. K4580-01, K4580-40)
• General cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. No. K4595-01, K4595-40)
• Fast growth: Mach1™-T1R Chemically Competent E. Coli (Cat. No. K4530-20)
• Provide your own: for flexibility and to save money (Cat. No. 450030)

We also offer a version of the kit that includes a PureLink™ Quick Plasmid Miniprep Kit (Cat. No. K4575-02) for use in isolation of clean, sequencing-ready, recombinant plasmid.

Related Links

• Custom Vector Construction and Cloning Services
• Plasmid DNA Purification Kit Selection Guide
• PCR Reagents, Instruments, and Supplies