5 min TOPO-Blunt Cloning Kit is the third generation TOPO cloning kit, which contains Topoisomerase, vector and self-ligation inhibitory factor. The kit utilizes a new type of Topoisomerase, with the optimal buffer, the enzyme activity is higher, and the high-efficiency connection can be completed in 5 minutes. The addition of a vector self-ligation inhibitory factor increases the positive rate of cloning. This kit is suitable for blunt-end cloning.
Fig 1. 5 min TOPO-Blunt Cloning Kit
Product Name: 5 min TOPO-Blunt Cloning Kit
Size: 20 rxns/40 rxns
|Components||C602-01 (20 rxns)||C602-02 (40 rxns)|
|5 × TOPO-Blunt Cloning Mixa||40 µl||80 µl|
|500 bp Control insert (20 ng/μl)||10 µl||20 µl|
|M13 Primer Mix (10 μM)b||200 µl||400 µl|
a. Contains Topoisomerase and pCE3 Blunt Vector (Amp resistance vector).
b. Contains M13 Forward Primer and M13 Reverse Primer.
Molecular cloning is an excellent technique for creating DNA-based experimental tools for expression in bacterial or mammalian cells1. Since this technology was invented, it has been widely used in various molecular biology experiments.
Topoisomerase Cloning (TOPO-Cloning) is a method that utilizes topoisomerase which has both restriction enzyme and ligase properties. It can specifically recognize the pentanucleotide sequence 5'-(C/T)CCTT-3', and can form a covalent bond with the phosphate group attached to 3´thymidine2. Topoisomerase is capable of cleaving a strand of DNA and unwinding it2. The 5'-OH at each end of the PCR product can attack the phosphotyrosine bond between the vector DNA and topoisomerase, thereby releasing the topoisomerase molecule and producing a double-nicked circular recombinant molecule (Fig 3). Subsequently, topoisomerase reconnects the cleaved strands. To take advantage of the religation activity of topoisomerases, we provide a linearized TOPO vector, the pCE3 Blunt Vector (Fig 2).
Fig 2. The sequence of pCE3 Blunt Vector | M13 Reverse primer site: bases 60 - 76; M13 Forward primer site: bases 209 - 225; Ampicillin resistance ORF (C): bases 226 - 1,086; pUC orih=gin: bases 1,210 - 1,798; (C): complementary strand
Advantages of TOPO-Cloning
1. No need for restriction digestions
2. No need to gel extract PCR products unless there are more than one band
3. Extremely efficient ligation
4. Less of vector self-ligation thus increasing the positive rate
5. Suitable for both blunt-ended and sticky-ended cloning
5 min TOPO-Blunt Cloning Kit (Vazyme #C602) is a product designed for blunt-ended cloning with all the advantages of TOPO cloning.
Fig 3. Workflow of 5 min TOPO-Blunt Cloning Kit:
(a). Add the amplification product (blunt-end) of a high-fidelity enzyme (such as Vazyme's Phanta series: https://www.vazymebiotech.com/pcr) into 5 × TOPO-Blunt Cloning Mix, and react at 20 ~ 37 °C for 5 min.
(b). The blunt-ended product 5'-OH attacks the phosphate bond between the topoisomerase and the vector, the topoisomerase is released. The vector and the blunt-ended product form a circular recombinant.
Ultra-high Positive Rate
In order to test the ligation efficiency of C602, the samples Blunt-1K, Blunt-3K, Blunt-7K and Blunt-10K were used for cloning reaction in PCR machine respectively. The obtained PCR products were purified and run on a 1% agarose gel for detection and identification. Similarly, to test the impurity tolerance of C602, samples Blunt-1K and Blunt-10K were treated in the same way. However, the PCR product was directly run on 1% agarose gel without purification.
Fig 4. The positive rate of C602 after a).ligation efficiency test and b).impurity tolerance test.
In conclusion, C602 has an ultra-high positive rate in both ligation efficiency test and impurity tolerance test.
The compatibility of ligation time of C602 was tested by gel recovery of purified fragments Blunt-1K (74.5 ng/μL) and Blunt-7K (157.65 ng/μL) from λDNA.
Set the connection time as 5 min, 10 min, 20 min and 30 min, respectively. Then, the ligation solution was transformed into competent cells, and the number of colonies was counted after incubation. The results are shown in Table 1.
Table 1. Colones counting after transformation: Studying the effect of ligation time on efficiency
In conclusion, extending the reaction time does not significantly affect the ligation efficiency of C602, the recommended ligation time is 5 min.
Freeze-thaw stability: C602 was repeatedly freeze-thawed in liquid nitrogen for 0, 5, 10, 15, and 20 times, and compared with the unfreeze-thawed control group. The freeze-thawed and unfrozen-thawed products were connected to Blunt-500bp and Blunt-2K respectively, count the number of single clones and test the positive rate.
Storage stability at 4°C: Store C602 at 4°C for 0, 3, 10, 14, 21, and 31 d, test its efficiency in connecting Blunt-1k and Blunt-3k, count the number of single clones, and detect the positive rate of C602 stored at 0 d and 31 d .
1) Number of single clones: Compared with the control group, if the deviation of the number of monoclonal reagents in the treatment group is within ±20%, it is judged that there is no significant difference, and the performance is stable.
2) Positive rate: Colony PCR test, the number of positive clones/total number of clones ≥ 14/16 can be judged as qualified and the positive rate is not significantly affected.
Figure 5. a).Statistics of colony count after repeated freezing and thawing; b).Statistics of colony count after storing at 4℃; c).Positive rate after repeated freezing and thawing; d).Positive rate after storing at 4℃
In conclusion, C602 has stable performance under repeated freezing and thawing conditions. After storing at 4°C for 3 days, its performance does not decrease significantly. However, it is recommended that the product be returned to -20°C in time after use.
Vazyme’s Cloning Products
Since the establishment in 2012, Vazyme has been dedicated to our mission "Science and Technology Make a Healthier Life" to focus on technology innovation and continuously expand the application fields of core technologies in life science. As a R&D based company, we have been holding ourselves to the highest standards of ethics, accountability and professionalism. Now, we have a variety of product lines based on our self-developed technologies.
The following are other cloning products that we can offer you (click on the Cat.No. to see the detail of each product):
|TOPO Cloning||5 min TA/Blunt-Zero Cloning Kit||
25 rxn / 50 rxn
|Fast Cloning||ClonExpress II One Step Cloning Kit||25 rxn / 50 rxn||C112-01/02|
|ClonExpress MultiS One Step Cloning Kit||10 rxn / 25 rxn||C113-01/02|
|ClonExpress Ultra One Step Cloning Kit||25 rxn / 50 rxn||C115-01/02|
|Fast Site-Directed Mutagenesis||Mut Express II Fast Mutagenesis Kit V2||
10 rxn / 25 rxn
|Mut Express MultiS Fast Mutagenesis Kit V2||
10 rxn / 25 rxn
|Traditional/TA Cloning||T4 DNA Ligase||40,000 U||C301-01|
|5 min Universal Ligation Mix||
50 rxn / 100 rxn
1. Lessard, J. C. (2013). Molecular cloning. In Methods in Enzymology (Vol. 529, pp. 85-98). Academic Press.
2. TOPO Cloning Technology. (n.d.). Retrieved from https://www.thermofisher.cn/cn/zh/home/life-science/cloning/topo/topo-resources/the-technology-behind-topo-cloning.html