The Zero Blunt® TOPO® PCR Cloning Kit for Sequencing provides a highly efficient, 5-minute, one-step cloning strategy for the direct insertion of proofreading-polymerase–amplified, blunt-ended PCR products into a plasmid vector for sequencing. Each kit uses the pCR™4Blunt-TOPO® vector (see figure) 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.
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. 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, which saves you 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 Kit for Sequencing—kit formats 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:
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