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TOPcloner™ Blunt Kit

No Product Name Cat.# Conc Size Price(₩) Add
1 TOPcloner™ Blunt Kit EZ002S - 20 reactions ₩ 250,000 추가
2 TOPcloner™ Blunt Kit EZ002M - 40 reactions ₩ 450,000 추가
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Product description

TOPcloner™ Blunt kit is suitable for cloning Pfu polymerase-amplified PCR products with blunt ends at room temperature in 5 min. The vector provided in this kit contains topoisomerase covalently coupled to its ends, which allows efficient cloning of blunt-ended DNA fragments to the vector without using a regular ligase.

 

Characteristics

 - Short reaction time (5 min)

 - Highly efficient cloning

 - Direct use of PCR products

 

Applications

 - Fast and efficient cloning of blunt-ended PCR products (for example, Pfu-amplified DNA)

 

Supplied with

- pTOP Blunt V2

- 6X TOPcloner™ Buffer

- 2X TOPsimple ™ DyeMIX-Tenuto

- DH5α Chemically Competent E. coli

- M13F, M13R (10 pmol/μl)

- Control insert DNA (20 ng/μl)

- Control primers (10 pmol/μl each)

- Sterile water

 

Quality control

 - Cloning efficiency: 99%

 

Standard reaction conditions

6X TOPcloner™ Buffer

1 μl

pTOP Blunt V2 (10 ng/μl)

1 μl

PCR product

1~4 μl

Sterile water

up to 6 μl

→ Incubate at room temperature(25) for 5 min

→ Transformation


Cautions
- The 5’ ends of PCR primers used for amplification of target DNAs to be cloned should not have 

  a 5' phosphate group.

- Purification of PCR products is recommended when smearing, multiple bands, and/or primer-dimers are 
  prominently observed or when PCR products are longer than 1 kb.
- Since non-specific PCR products shorter than 100 bp can be ligated to the TOPcloner vector with high 
  efficiency,  they can severely reduce cloning efficiency of target DNA. In this situation, it is recommended to
  purify the PCR products in order to remove nonspecifically amplified short DNA.



Figure. A schematic map of pTOP Blunt V2 (3807 bp).

A molecule of topoisomerase (TOPO) is covalently coupled to the 3' blunt duplex ends (nt positions 294 and 295) of pTOP Blunt V2 (Cat.# EZ002 and EZ012). Therefore, pTOP Blunt V2 can form a covalent bond with blunt-ended PCR products obtained by Pfu DNA polymerase.

 - Lac promoter/operator : 95-216
 - M13 Reverse Primer binding site : 205-221
 - LacZ ORF : 217-534
 - MCS, Multiple Cloning Sites : 234-357
 - M13 (-20) Forward Primer binding site : 391-406
 - ccdB ORF : 544-846
 - Kanr gene : 1057-1989
 - Ampr gene : 2007-2867
 - pUC origin : 3012-3685


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