What is TA cloning used for?
Abstract. TA cloning is one of the simplest and most efficient methods for the cloning of PCR products. The procedure exploits the terminal transferase activity of certain thermophilic DNA polymerases, including Thermus aquaticus (Taq) polymerase.
What is a TA vector?
T vectors are linearized plasmids that have been treated to add T overhangs to match the A overhangs of the PCR product. PCR fragments that contain an A overhang can be directly ligated to these T-tailed plasmid vectors with no need for further enzymatic treatment other than the action of T4 DNA ligase.
Is Topo cloning directional?
Directional TOPO cloning enables cloning of blunt-ended PCR products in a 5´→3´ orientation directly into a expression vector using a 5-minute ligation reaction, thereby eliminating subcloning steps and saving you time.
What is a TA cloning vector?
T-vector cloning, or TA cloning, is a convenient method for cloning PCR products generated with Taq DNA Polymerase. The pGEM®-T vectors are a popular choice for general PCR cloning.
What is end repair and dA tailing?
CD End Repair/dA-Tailing Module for Illumina uses a one-step reaction to combine end-repair, 5′ phosphorylated, and dA-tailing to convert fragmented DNA into 5´-phosphorylated and 3´-dAtailed DNA fragments enabling direct ligation of Illumina sequencing adapters.
How much DNA is needed to TOPO clone?
50-100ng
For transformation you should not use more than 50-100ng total DNA. Before TOPO cloning, you should purify the PCR product from enzymes, buffers and primers (spin column or PEG precipitation).
Is Gateway cloning expensive?
Gateway cloning does take more time for initial set-up, and is more expensive than traditional restriction enzyme and ligase-based cloning methods, but it saves time, and offers simpler and highly efficient cloning for down-stream applications.
Is Gateway cloning reversible?
Invitrogen Gateway recombination cloning uses a one hour reversible recombination reaction, without using restriction enzymes, ligase, subcloning steps, or screening of countless colonies, thereby saving you time, money, and effort.
What is da tailing?
Tailing is an enzymatic method for adding a non-templated nucleotide to the 3′ end of a blunt, double-stranded DNA molecule. Tailing is typically done to prepare a T-vector for use in TA cloning or to A-tail a PCR product produced by a high-fidelity polymerase (not Taq) for use in TA cloning.
What is a tailing in library prep?
After fragmentation, the DNA fragments are end repaired or end polished. Generally, a single adenine base is added to form an overhang via an A-tailing reaction. This A overhang allows adapters containing a single thymine overhanging base to base pair with the DNA fragments.
How does the TA Cloning Kit work?
The TA Cloning® Kit uses the pCR™2.1 cloning vector and ExpressLink™ T4 DNA Ligase to generate a ligation product in a fifteen-minute, room-temperature ligation step. Reactions typically yield >80% recombinants containing inserts.
What competent cells are available in Tata cloning kits?
TA Cloning kits are available without competent cells ( K2020-20 and K2020-40) or with Invitrogen One Shot INVαF’ Chemically Competent E. coli ( K2000-01 and K2000-40 ), and One Shot TOP10F’ Chemically Competent E. coli ( K2040-01 and K2040-40 ) in both 20 and 40-reaction pack sizes.
What are the available vector (dual promoter) kits for TA cloning?
For TA Cloning with the pCR II Vector (dual promoter) kits are available without competent cells ( K2750-20 and K2750-40 ), with One Shot INVαF’ Chemically Competent E. coli ( K2050-01 and K2050-40 ), and One Shot TOP10F’ Chemically Competent E. coli ( K2060-01 and K2060-40) in both 20 and 40-reaction pack sizes.
How long does it take to ligation a TA kit?
With the addition of the Invitrogen ExpressLink T4 DNA Ligase into TA Cloning Kits, ligation can be performed at room temperature in only 15 minutes with reactions typically yielding >80% recombinants containing inserts. TA Cloning kits are available with a choice of Invitrogen pCR 2.1 and pCR II vectors.