I'm looking for specific procedure of TA cloning. Best if someone provides this with published article.
Hi Mr Niloy Kumar
at first you can read our article "Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods ".
The procedure of TA cloning is:
Terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase adds a single adenosine to the 3'-ends of a double stranded DNA molecule so, most PCR fragments which amplified by non proofreading thermostable DNA polymerases like Taq polymeras The use of a linearized “T-vector” which has single 3' products, facilitated by complementarily between the PCR product 3' This method requires the use of T4 DNA ligase to covalently link the compatible ends of the DNA fragment and the linearized plasmid to forming a single cyclic molecule that is capable of autonomous replication in host component E.coli.
Here is a link to the company website where you can buy a TA cloning kit. Read the product manual and it will tell you what you need to know.
https://www.thermofisher.com/us/en/home/life-science/cloning/topo/topo-ta-cloning.html
this will also help while using not fresh/old PCR product
http://www.protocol-online.org/prot/Protocols/Adding-3--A-overhang-to-a-PCR-product-3436.html
https://openwetware.org/wiki/Addition_of_3%27_A_overhangs_to_PCR_products
- Badges
- Science topic
- Similar topics
- Linguistics
- Composition Studies
- Publisher