Protocol: Preparing T-vector using Terminal Transferase and ddTTP

T-vectors are linearized vectors that contain a single T-overhang at the 3’-end of both DNA strands. T-vectors are useful for cloning of PCR amplified DNA fragments. PCR reactions which use non-proofreading Taq DNA polymerase have an extra single nucleotide mostly adenine (A) at the 3’-end of amplified DNA fragments. The A overhang of the PCR-amplified DNA fragment pairs with the T overhang of the T-vector (similar to sticky end cloning), and subsequent ligation of their ends by ligase enzyme results in a circular recombinant DNA molecule.

T-vectors can be created by several methods. Restriction enzymes that can generate 3’- single T-overhang upon cleavage can be used to generate T-vectors. Non-proofreading Taq DNA polymerase and terminal transferase (also known as terminal deoxynucleotidyl transferase) can also be used to add a single  3’- T-overhang to the blunt-end linearized vectors.

Terminal transferase is a non-template DNA polymerase that can elongate 3’ end of DNA without the need of a template. Since T-vectors require addition of a single T at 3’-end, reaction is performed in presence of dideoxyTTP. Due to dideoxyTTP (ddTTP), after addition of a T at 3’ end, no further addition can be made by enzyme.

Here we describe a method that uses Terminal transferase and ddTTP to add a single 3’- T-overhang of both strands of the blunt-end linearized vector. The plasmid vector is digested with a restriction enzyme that generates blunt-end linearized vectors. Linearized vector is purified and subjected to terminal transferase reaction in presence of ddTTP. After the reaction, DNA is again purified and stored in the freezer (-20°C or -80°C) until use.

REQUIREMENTS

Reagents and solutions
Plasmid vector (e.g., pBlueScript)
Restriction enzyme (e.g., EcoRV or SmaI)
Terminal transferase enzyme and buffers (available from commercial suppliers)
ddTTP

Equipment and disposables
Water bath set at 37 °C /Heat block set at 37°C
Eppendorf tube (1.5 ml or 0.5 ml, Nuclease-free)
Micropipette and tips

PROCEDURE

Note: Here we describe this method by using an example of pBlueScript. pBlueScript vector contains a single EcoRV restriction site in the multiple cloning site. Digestion of pBlueScript with EcoRV generates linear DNA with blunt ends. Digested plasmid is purified (preferably gel elution) and incubated with Terminal  transferase enzyme in presence of dideoxyTTP under suitable reaction conditions. After 60 min of incubation, the enzyme reaction is stopped by heat inactivation and purified again.

Step 1: Digest the plasmid vector with a blunt-end cutter restriction enzyme that has a single restriction site within a multiple cloning site.
For example, you can use either EcoRV or SmaI restriction enzyme to digest 5 – 10 µg of pBlueScript plasmid vector to linearize it. Use the regular protocol for digestion as suggested by the restriction enzyme supplier. A 50 – 100 µl final reaction volume is recommended to digest 5 – 10 µg plasmid DNA. Digesting must be 100% complete with no undigested circular plasmid in the reaction. To make sure that the digestion of plasmid DNA is 100% complete, you can run the digested plasmid on agarose gel and check whether all the plasmid DNA are linearized without any traces of supercoiled and nicked circular form.

Step 2: Purify the digested blunt-ended DNA using an appropriate procedure
There are several methods and commercial kits are available that can be used for purification of plasmid DNA. We recommend running all the digested plasmid in agarose gel and eluting the linearized plasmid DNA band from agarose gel. This will ensure that linearized plasmid DNA is not contaminated with undigested plasmid and reduce the incident of false positives when you use this T-vector for cloning. Purified DNA must be dissolved or eluted in water instead of TE buffer. 

Step 3: Use purified blunt-ended DNA for terminal transferase reaction.
Incubate the purified blunt-ended plasmid DNA with terminal transferase and ddTTP in reaction condition as described in the supplier’s manual.

Step 4: After the terminal transferase reaction is over, inactivate the enzyme and purify the T-vector by phenol:chloroform extraction or any suitable commercial kits.

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