TA cloning exploits the property of nonproofreading thermostable DNA polymerases (such as Taq polymerase) to add an extra “A” nucleotide to each 3′-end of amplified DNA fragments. T-vectors are linearized vectors that have an unpaired T nucleotide at the 3’-end of each strand. The pairing of “A” overhang of PCR amplified DNA fragment to “T” overhang of T vector and subsequent ligation results in a recombinant DNA molecule.
REQUIREMENTS
Reagents and solutions
DNA fragments (PCR -amplified using non-proofreading Taq DNA polymerase)
T-vector (self made/commercially available)
T4 DNA ligase
10x T4 DNA ligase reaction buffer supplemented with ATP
High efficiency competent cells (E. coli DH5a, Top10)
Appropriate antibiotic (Ampicillin for pBluescript)
LB medium (LB agar plate with appropriate antibiotics)
SOB medium
Equipment and disposables
Micropipette and tips
Microcentrifuge tube (0.5 ml or 1.5 ml)
Water bath
PROCEDURE
Step 1: Purify PCR-amplified DNA fragments using an appropriate method.
We recommend Gel purification of PCR-amplified DNA fragments. Gel purification requires running PCR product in an agarose gel and subsequently dissecting out a piece of agarose gel that contains expected size DNA fragments. From this piece of gel, DNA fragments can be extracted by several methods including commercially available gel elution kits. Purified DNA should be dissolved in water. Determine the concentration of DNA fragments using nanodrop or any other methods.
Step 2: Set the ligation reaction and incubate at 14°C for 4 h – overnight.
Reaction Components | Fragment – vector ligation reaction | Control ligation reaction ¶ | ||
Final concentration | Amount taken (Ligation reaction) | Final concentration | Amount taken (Ligation reaction) | |
10x ligation buffer (supplemented with ATP) | 1 x | 1 µl | 1 x | 1 µl |
T-vector (≈4000 bp) 25 ng/µl | 5 ng/µl | 2 µl | 5 ng/µl | 2 µl |
Purified DNA fragment (≈1000 bp) 20 ng/µl | 2 – 4 ng/µl | 1-2 µl | 0 | 0 |
Water | to a total volume of 9 µl * | to a total volume of 9 µl * | ||
The T-vector:DNA fragment molar ratio should be between 1:1 to 1:3. ¶ Control ligation helps you to determine the self ligation efficiency of a T-vector. It is recommended to set the self ligation of the T-vector if you have prepared the T-vector yourself. * Adjust the water amount according to the concentration of your DNA insert. | ||||
Now add 1 µl T4 DNA ligase (stock conc 5 U/µl) in each reaction mix and incubate at 14°C for 4 h – overnight.
Step 3: Transform competent cells with all ligation reaction mix.
Use regular protocol to transform E. coli competent cells with ligation mixture. You must choose a suitable E. coli strain. After transformation, plate the bacterial cells on a LB-agar plate containing appropriate antibiotics.
Screen some colonies for the presence of cloned DNA in T-vector.
