Ligation and Transformation
Ligate the PCR product into a plasmid and transform bacteria. Students directly clone their PCR products into the pJET1.2 vector and immediately transform bacteria using a protocol that takes less than 2 hours to go from purified PCR product to transformed bacteria plated on agar.
Students first remove the 3'-dA overhang from their PCR product which results from amplification with Taq polymerase using a proofreading polymerase. A 10 minute protocol is then used to ligate the blunted PCR product into a pre-opened and blunted vector — pJET1.2. The pJET1.2 plasmid positively selects for successful insertion of fragments because the cloned fragment inserts into a lethal gene present in the plasmid that prevents its activation. Bacteria that are transformed with re-ligated vector activate the gene and are killed. This results in higher transformation efficiency than traditional blue-white cloning. Bgl II restriction sites are located on either side of the pJET1.2 cloning site, which allow students to determine if their gene of interest was successfully ligated once they have isolated candidate plasmids.
To transform the ligation into bacteria, students make competent cells by inoculating specialized growth media and performing a series of microcentrifugations and washes in a specialized transformation buffer. Competent cells are then added to the ligations on ice and plated directly on warm agar plates. Between 10 and 100 colonies should be expected from transformed ligations with the Cloning and Sequencing Explorer Series.
