Cloning for Sequencing Applications

The TOPO® cloning kits for sequencing allow fast cloning and streamlined sequencing of PCR products. These kits contain TOPO® cloning vectors with a minimized multiple cloning site that positions the T7 and T3 priming sites only 33 bp away from the PCR product insertion site (Figure 1). This means you’ll sequence more of your insert and less of the vector.

Choice of Vector

The pCR®4-TOPO® vector supplied in the TOPO® TA Cloning® Kit for Sequencing has 3´-T overhangs for cloning Taq-amplified PCR products. The pCR®4Blunt-TOPO® vector supplied in the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing has blunt ends for cloning PCR products amplified with proofreading polymerases.  Convenient features of these sequencing vectors include:

  • T7 and T3 promoter/priming sites for sequencing and in vitro transcription/translation
  • M13 forward (–20) and reverse priming sites for sequencing or PCR screening
  • The ccdB gene to eliminate background and improve results    
  • EcoR I sites flanking the PCR product insertion site for easy removal of inserts
  • Unique Sse8387 I site to produce nested deletions for sequencing internal regions of your insert
  • Kanamycin and ampicillin resistance genes for your choice of selection in E. coli

Figure 1:  pCR® 4-TOPO® cloning vectors for sequencing.


Maximize transformation

Both kits are offered with Mach1™-T1R competent cells for the fastest cloning. The Mach1™-T1R E. coli strain is the fastest-growing cloning strain, allowing you to achieve sufficient growth for minipreps from an overnight colony in just four hours, which saves an entire day over most other cloning strains. We also offer both TOPO® TA Cloning® Kits for sequencing with our highly popular TOP10F’ or MAX Efficiency® DH5α™-T1 Phage-Resistant competent cells.