TOPO® TA Cloning® Kit for Subcloning, with One Shot® TOP10 chemically competent E. coli cells

Catalog number: K450001

Invitrogen™  Related applications: PCR Cloning

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Description

TOPO® TA Cloning® Kits for Subcloning 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 subcloning. Each kit uses the pCR™ 2.1-TOPO® TA vector with convenient restriction sites for subcloning. The cloning kits are available with a variety of competent cells, or without competent cells, depending upon your needs and budget. Features of the TOPO® TA Cloning® Kits for Subcloning:

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
Simple—no ligase, post-PCR procedures, or PCR primers containing specific sequences are required

TOPO® TA Cloning® Kits for Subcloning—overview

Vector: pCR™ 2.1-TOPO® TA vector—subcloning vector with 15 convenient restriction sites flanking your insert for easy, directional subcloning

Cloning method: TOPO® TA Cloning® —Topoisomerase I-based, 5-minute ligation of PCR products with 3′-A overhangs (Taq polymerase amplified) to TOPO® vector with T overhangs

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

pCR™ 2.1-TOPO® TA vector—cloning and subcloning simplicity
The pCR™ 2.1-TOPO® TA vector is linearized with 3'-thymidine (T) overhangs for direct ligation of Taq polymerase-amplified PCR products (TA cloning®) and is "activated" with covalently bound Topoisomerase I. There is no need to add ligase and cloning is complete in 5 minutes. EcoRI sites flanking the PCR product insertion site allow for easy excision of inserts or use any combination of 15 convenient restriction sites flanking your PCR insert for easy, directional subcloning.

pCR™ 2.1-TOPO® TA clone selection and manipulation
The pCR™ 2.1-TOPO® TA vector contains both ampicillin and kanamycin resistance markers and the LacZα gene for blue/white screening.

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 E. coli competent cells.

Efficient cloning
With up to 95% of clones carrying the desired insert, you can screen fewer clones, saving time and money. The pCR™ 2.1-TOPO® TA vector used in this kit comes with 3'-T overhangs for efficient ligation of Taq polymerase-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 Subcloning—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 cells (Cat. No. K4500-01, K4500-40)
• High-efficiency cloning: TOP10 Electrocomp™ Cells (Cat. No. K4560-01, K4560-40)
• General cloning, bacteriophage T1 resistance: DH5α-T1R cells (Cat. No. K4520-01, K4520-40)
• Fast growth: Mach1™ -T1R Chemically Competent E. Coli (Cat. No. K4510-20)
• Provide your own: for flexibility and to save money (Cat. No. 450641)

We also offer two versions of the kit that include a PureLink™ Quick Plasmid Miniprep Kit (Cat. No. K4500-02 and K4510-02) for use in isolation of clean, sequencing-ready, recombinant plasmid.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Vector Type: TOPO-TA Cloning Vectors
Cloning Method: TOPO®-TA
Bacterial or Yeast Strain: TOP10
Product Size: 25 reactions

Contents & storage

Box 1:
• Topoisomerase I-activated pCR™2.1-TOPO® vector
• PCR buffer
• Salt solution
• dNTPs
• Control template
• M13 forward and reverse primers
• Control PCR primers
• Sterile water

Store at -5 to -30°C.
All reagents are stable for 6 months when properly stored.

Box 2:
• One Shot® Chemically Competent or Electrocomp™ E. coli
• S.O.C. medium
• Supercoiled pUC19 control plasmid

Store at -68 to -85°C.

Documents

Manuals & protocols

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