Ligation and Transformation Module

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Overview

Lab Preparation Checklist
Kit contains sufficient materials for 12 workstations.
T4 DNA ligase, 10 µl 1
2x ligase buffer, 100 µl 1
Proofreading DNA polymerase, 10 µl 1
Cloning vector, 10 µl 1
Bgl II restriction enzyme, 50 µl 1
10x restriction enzyme buffer, 100 µl 1
C-Growth medium, 20 ml 1
Transformation A solution, 5 ml 1
Transformation B solution, 5 ml 1
IPTG, 0.1 ml 1
Sterile water, 2.5 ml 1
Colored micro test tubes, 2.0 ml 120
Curriculum, including Teacher's Guide, Student Manual, and graphic Quick Guide 1
Required Accessories Not Included in Kit:
Water bath
Microcentrifuge
Incubation oven
Shaking water bath

Note: Bacterial culturing reagents such as LB broth and LB agar are contained in the Microbial Culturing module.

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.

The ligation and transformation module is an integral component of the Cloning and Sequencing Explorer Series. Use this module to directly ligate PCR products into the pJET1.2 plasmid vector and immediately transform freshly prepared competent bacteria with the ligation reaction. Competent cells are then plated directly on warm agar plates and incubated overnight at 37°C.

Once transformed bacteria have been propagated, plasmid DNA can be isolated and digested with BglII enzyme. The BglII restriction sites located on either side of the pJET1.2 cloning site allow students to determine if their PCR product was successfully ligated.

This module may also be purchased separately and used to clone any PCR product. The entire ligation and transformation protocol takes approximately 2 hours to complete and does not require commercial competent cells, a refrigerated microcentrifuge, or a –80°C freezer, making it the method of choice for educators worldwide.

Ligation and Transformation Module #166-5015EDU
Ligation And Transformation Module

166-5015EDU
Fifth module in Cloning and Sequencing Explorer Series classroom study kit, includes T4 ligase, cloning vector, growth medium, curriculum, and more, for 12 workstations; education use only

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