Team:Aberdeen Scotland/Cloning/

Notes about Cloning DNA

Molecular Cloning of DNA involves a number of steps which can be broadly categorized as: Restriction Digestion (cutting of DNA using restriction enzymes); Ligation (joining of DNA using DNA ligases) and Transformation (propogation and selection of recombinant DNA inside a living cell). Since DNA Digestion and Transformation are covered separately this page simply outlines the protocol used for Ligation.

Roche: Rapid DNA Ligation Kit

Preparations:
Prepare 1x conc. DNA dilution buffer.
Prepare a total of 30-50 ng of DNA using a 1:3 molar ratio of vector : insert.

1. Dissolve Vector DNA and Insert DNA, in carefully mixed DNA dilution buffer.
2. Carefully mix T4 DNA Ligation buffer and subsequently add 10ul of DNA ligation buffer to mixture.
3. Add 1ul of T4 DNA Ligase to the mixture.
4. Incubate mixture for 5 minutes at 15-25*C
6. Store Ligation Mixture WITHOUT Heat Inactivating the Enzyme at -15 to -25 *C

Protocols

Competency