컨텐츠 바로가기 영역
주메뉴로 바로가기
본문으로 바로가기

All products

Home > PRODUCTS > All products

TOPcloner™ TA Kit

No Product Name Cat.# Conc Size Price(₩) Add
1 TOPcloner™ TA Kit EZ001S - 20 reactions ₩ 250,000 추가
2 TOPcloner™ TA Kit EZ001M - 40 reactions ₩ 450,000 추가
Product Name Cat.# Size Price(₩) EA Total(₩) 삭제
Total : 장바구니에 담기 바로 구매 하기
plus minus 삭제

Product description

TOPcloner™ TA kit is suitable for cloning Taq polymerase-amplified PCR products with A-tail overhangs at room temperature in 5 min. The vector provided in this kit contains topoisomerase covalently coupled to its ends, which allows efficient cloning of DNA fragments with A-tail overhangs 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 A-tailed PCR products (for example, Taq-amplified DNA)

 

Supplied with

- pTOP TA V2

- 6X TOPcloner™ Buffer

- 2X TOPsimple ™ DyeMIX-Tenuto

- M13F, M13R (10 pmol/μl)

- Control insert DNA (20 ng/μl)

- Control primers (10 pmol/μl each)

- DH5α Chemically Competent E. coli

- Sterile water

 

Quality control

- Cloning efficiency: 99%

 

Standard reaction conditions

6X TOPcloner™ Buffer

1 μl

pTOP TA 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.
- We recommend using TOPcloner Blunt kit (Cat.# EZ002) for cloning DNA fragments amplified with Pfu 
  DNA polymerase. Alternatively, one unit of Taq polymerase can be added to the Pfu amplification reaction, 
  followed by incubation at 72℃ for 10 min. This allows Taq polymerase to introduce A-tails at the blunt 
  ends of PCR products obtained with Pfu. The DNA products formed this way can be cloned into pTOP TA V2.
- Note that the addition of A-tails is most efficient for DNA fragments with C as a terminal residue, but most 
  inefficient with A as a terminal residue. This should be taken into considerations in designing oligonucleotides 

  or PCR primers.




Figure. A schematic map of pTOP TA V2 (3807 bp).
 A molecule of topoisomerase (TOPO) is covalently coupled 
to each of the two 3' one-nt protruding ends (nt positions 294 and 295) of pTOP TA V2 (Cat.# EZ001 and EZ011). Therefore, pTOP TA V2 is ready to form a covalent bond when mixed with PCR products with one-nt 3' A overhang. The addition of one A to the duplex DNA ends is intrinsic to Taq 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


List