Zero Blunt® TOPO® Cloning Kit for Sequencing with One Shot® TOP10 Chemically Competent E. coli by Thermo Fisher Scientific Invitrogen

The Zero Blunt® TOPO® PCR Cloning Kits for Sequencing provide a highly efficient, 5 minute, one-step cloning strategy ("TOPO® Cloning") for the direct insertion of proofreading-polymerase–amplified blunt-end PCR products into a plasmid vector for sequencing. Each kit uses the pCR™4Blunt-TOPO® vector with specially designed sequencing primer sites that return more insert sequence and less vector sequence from each reaction.

These kits include everything necessary to clone and select recombinant vectors containing your PCR fragment of choice.

• Get More Sequence—Allows for more insert sequence and less vector sequence when using standard sequencing primers
• ast and Easy—Go from PCR-to-clones in just 3 steps and in as little as 5 minutes hands-on time
• Efficient—Achieve up to 95% clones with correct insert
• Proven—Reliable performance for over a decade with over 4000 citations

Zero Blunt® TOPO® Cloning Kits for Sequencing Kit Overview

VECTOR: pCR™4Blunt-TOPO® Vector

• Optimized cloning vector for improved sequencing results

CLONING METHOD: TOPO® Cloning

• Topoisomerase I based 5 minute ligation of proofreading-polymerase-amplified PCR products with blunt ends to the vector

COMPETENT CELLS: Various Options

• Choose from kits with either general, high-efficiency, bacteriophage T1-resistant, or fast-growing competent cells

pCR™4Blunt-TOPO® Vector—Optimized for Sequencing

We have removed much of the multiple cloning site from the pCR™4Blunt-TOPO® 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™4Blunt TOPO® vector has sites for 4 common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.

pCR™4Blunt-TOPO® Clone Selection and Manipulation

The pCR™4Blunt-TOPO® 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 the provided E. coli competent cells.

Efficient Cloning

With up to 95% of clones carrying the desired insert, you can screen fewer clones to help you save time and money. The pCR™4Blunt-TOPO® vector used in this kit comes with no overhangs for efficient ligation of PCR products created by proofreading, thermostable polymerases that leave blunt-ended PCR products.

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 Kits for Sequencing Kit Options

The Zero Blunt® TOPO® PCR Cloning Kit for Sequencing can be purchased with a variety of competent cells that deliver different advantages depending on what your needs are:

• General cloning: TOP10 (Cat. No. K2875-J10, K2875-20, K2875-40)
• High efficiency cloning: TOP10 Electrocomp™ Cells (Cat. No. K2880-20, K2880-40)
• eneral cloning, bacteriophage T1 resistance: DH5α-T1R (Cat. No. K2895-20)
• Fast growth: Mach1™-T1R Chemically Competent E. Coli (Cat. No. K2835-20)

TOPO® products are For Research Use Only. Not intended for any animal or human therapeutic of diagnostic use.