Zero-background TA topoisomerase cloning kit for fast cloning of DNA fragments up to 10 kb generated by Taq DNA polymerase (3′-A overhang). Uses Vaccinia virus topoisomerase I strand-transfer activity for 5-minute ligation.
Key Features
- 5-minute room-temperature ligation (topoisomerase I)
- Zero-background vector system
- Clones fragments up to 10 kb with 3′-A overhangs
- Includes 1000 bp control insert for verification
Specifications
| Pack sizes | 20 rxns (CYCV1401) / 80 rxns (CYCV1402) |
| Components | pTOPO-T Vector (30 ng/µl), 1000 bp Control, 10× Enhancer |
| Storage | -20°C; stable for 12 months |
| Shipping | Ice packs; store at the listed temperature on arrival |
Typical Applications
- TA cloning of Taq PCR products with 3′-A overhangs, up to 10 kb.
- 5-minute room-temperature ligation; 1000 bp control insert is in the box.
- DNA must not be 5′-phosphorylated. Competent cells are not included.
pTOPO-TA FAQ
Can I clone proofreading (blunt-end) PCR products with the TA kit?
Usually no. High-fidelity / proofreading polymerases generate blunt-ended products without 3′ A-overhangs, so TA ligation needs an A-tail first. The manual instructs end polishing/tailoring the PCR product with Taq DNA polymerase before the TA reaction. Alternatively, skip the A-tailing step entirely and use our pTOPO-Blunt Kit (CYCV16), which clones blunt products directly.
What insert sizes does the manual support?
Insert fragments from 100 bp to 5 kb are demonstrated in the manual, with a design limit of up to 10 kb. Reaction time is 5 minutes at room temperature.
How are recombinants selected?
The vector uses zero-background selection — no blue-white screening is required. The manual does not state a numeric positive-clone rate.
Are competent cells included?
No — the kit contains the pTOPO-TA vector and reaction components only; competent cells and ligase are not included. You transform with your own cells.
Performance at a glance
| Insert size | Up to 10 kb (design limit); 100 bp – 5 kb demonstrated (10–150 ng) |
| Insert amount guide | 100–1000 bp → 10–40 ng; 1000–2000 bp → 40–80 ng; 2000–5000 bp → 80–150 ng |
| Reaction | 5 minutes at room temperature (20–30°C), 10 µl system |
| Selection | Zero background — no blue-white screening needed |
| Vector | 1865 bp, high-copy, pUC ori, AmpR |
| Flanking restriction sites | Pst I, Hind III, EcoR V, EcoR I, Not I |
| Preferred insert ends | 3′-A overhang from Taq PCR products; must NOT be 5′-phosphorylated |
| Storage | −20°C, stable 12 months |
| Pack sizes | 20 rxns (CYCV1401) / 80 rxns (CYCV1402) |
All values from the CYCV14 manual (PDF).
Limits and handling
- For laboratory research use only. Not for diagnostic or therapeutic use.
- Shipped with ice packs. Store at the temperature in the specifications table on arrival.
- Store at −20°C (manual: 12 months).
- DNA fragments must not be 5′-phosphorylated. TA (A-overhang) ends from Taq are preferred. Inserts up to 10 kb.
- Incubate the cloning reaction no more than 5 minutes at 20–30°C. Mix competent cells gently — do not pipette up and down.
Who sets these specifications
Changyu Bio manufactures Cat# CYCV14 and is responsible for the specifications on this page. Confirm pack sizes, storage, and protocol against the PDF manual before use. Quotes: market@changyubio.com or WhatsApp +86 18665577773.
Cat# CYCV14 — For research use only (RUO). Not for diagnostic or clinical use. Contact market@changyubio.com or WhatsApp +86 18665577773 for pricing and export delivery to Africa, Southeast Asia and worldwide.