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, and are available with a variety of competent cells, or no competent cells, depending on your needs and budget. These kits include everything necessary to clone and select recombinant vectors containing your PCR fragment of choice.
TOPO TA Cloning Kits for Sequencing: overview
Vector: pCR4-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 general, high-efficiency, bacteriophage T1-resistant, or fast-growing competent cells, or use your own
pCR4-TOPO TA vector: optimized for sequencing
We have removed much of the multiple cloning site from the pCR4-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 pCR4-TOPO TA vector has sites for 4 common sequencing primers: M13 forward, M13 reverse, T7, and T3. The kits include an aliquot of each.
pCR4-TOPO TA clone selection and manipulation
The pCR4-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.
With up to 95% of clones carrying the desired insert, you can screen less clones and save time and money. The pCR4-TOPO TA vector used in this kit comes with 3'T overhangs for efficient ligation of Taq-amplified PCR products, which contain 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 upon your needs:
- 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)
- Provide your own: for flexibility and to save money (Cat. No. 450030)
• Resistant to Ampicillin (AmpR) and Kanamycin (KanR)
• Get more sequence: allows for more insert sequence and less vector sequence when using standard sequencing primers
• Fast and easy: go from PCR to clones in just three 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
Cloning, PCR Cloning
Shipping Condition: Dry Ice
|Kit, Cloning for Sequencing|
|Cloning, PCR Cloning|
|Box 1: Topoisomerase I-activated pCR4-TOPO vector, PCR buffer, Salt solution, dNTPs, Control template, T3, T7, M13 forward, and M13 reverse primers, Control PCR primers, Sterile water|