Zero Blunt® TOPO® PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli (Supply Center Packaging)

Catalog number: K280020SC

Invitrogen™  Related applications: PCR Cloning

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Description

Zero Blunt® TOPO® PCR Cloning Kit provides a highly efficient, 5-minute, one-step cloning strategy ("TOPO® Cloning") for the direct insertion of blunt-ended PCR products amplified with proofreading thermostable polymerases into a plasmid vector. Each kit has the Zero Blunt® TOPO® vector (see figure) containing the ccdB gene for positive selection, only permitting growth of plasmid vectors with recombinants. Zero® Blunt® TOPO® kits are available with a variety of competent cells, or without competent cells, depending on your needs and budget. This product comes in Supply Center packaging, which features an additional outer box that holds both the TOPO® vector box and the competent cells box. This special packaging helps ensure that visitors to a Supply Center obtain the complete kit.

Features of the Zero® Blunt® TOPO® PCR Cloning Kits:

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.

Zero Blunt® TOPO® Cloning Kits—overview

Vector: pCR™Blunt II-TOPO® vector—contains the ccdB gene, allowing for direct selection of recombinants

Cloning method: TOPO® cloning—Topoisomerase I–based, 5-minute ligation of blunt-ended PCR products amplified with proofreading thermostable polymerases

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

pCR™Blunt II-TOPO® vector—designed for proofreading polymerases
The pCR™Blunt II-TOPO® vector is designed to clone blunt-ended PCR products generated by thermostable proofreading polymerases such as Platinum® Pfx DNA Polymerase. The pCR™Blunt II-TOPO® vector contains:

• EcoRI sites flanking the PCR product insertion site for easy excision of inserts
• Kanamycin and Zeocin™ resistance genes for your choice of selection in E. coli
• SP6 promoter/primer site for in vitro RNA transcription and sequencing
• M13 forward and reverse primer sites for sequencing or PCR screening

pCR™Blunt II-TOPO® clone selection
pCR™-Blunt II-TOPO® allows for direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. The vector contains the ccdB gene fused to the C-terminus of the LacZα fragment. Ligation of a blunt-end PCR product disrupts expression of the LacZα-ccdB gene fusion, permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required.

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 less clones, saving you time and money. The pCR™Blunt II-TOPO® vector used in this kit comes with lethal ccdB gene for efficient selection of blunt-ended PCR recombinants products generated bythermostable proofreading polymerases.

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.

Zero Blunt® TOPO® PCR Cloning® Kit options
The Zero Blunt® TOPO® PCR Cloning Kit for direct insertion of blunt-ended PCR products into a plasmid vector can be purchased with a variety of competent cells that deliver different advantages depending upon your needs:

General cloning: TOP10 cells (Cat. No. K2800-20, K2800-40)
High-efficiency cloning: TOP10 Electrocomp™ Cells (Cat. No. K2860-01, K2860-40)
General cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. No. K2820-20, K2820-40)
Fast growth: Mach1™-T1R Chemically Competent E. Coli (Cat. No. K2830-20)
Provide your own: for flexibility and to save money (Cat. No. 450254)

We also offer a version of the kit that includes a PureLink® Quick Plasmid Miniprep Kit (Cat. No. K2800-02) for use in isolation of clean, sequencing-ready, recombinant plasmid.
For Research Use Only. Not for use in diagnostic procedures.

Specifications

Vector Type: Blunt DNA Cloning Vectors
Cloning Method: Blunt TOPO®
Bacterial or Yeast Strain: TOP10
Product Size: 25 reactions
Shipping Condition: Dry Ice

Contents & storage

Box 1:
• Linearized and topoisomerase I-activated pCR™Blunt II-TOPO® vector
• Salt solution
• dNTPs
• Control template
• M13 forward and reverse 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.

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Manuals & protocols

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