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. 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 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—Achieve up to 95% clones with correct insert • Proven—Reliable performance for over a decade with over 4000 citations
TOPO® TA Cloning® Kits for Sequencing Kit 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 either general, high-efficiency, bacteriophage T1-resistant, or fast-growing competent cells
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 to help you 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 with 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 on what your needs are:
• 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)
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.
TOPO® products are For Research Use Only. Not intended for any animal or human therapeutic of diagnostic use.
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