Bio-Rad Ligation and Transformation Module - Manual (Page 36)

Instructor’s Advance Preparation
In the first part of this activity, students will insert (ligate) the purified PCR
products into the pJet1.2 blunted vector. They will then transform competent
bacteria with the ligation reaction mixture.
Note: Students can either proceed directly to transformation activity or
store the ligation reaction at 4°C or –20°C until the next laboratory session.
Note: In order to complete the laboratory more efficiently, preparation for
the transformation step can be initiated prior to performing the ligation
reaction, allowing immediate transformation of competent cells with the
products of the ligation reaction. Refer to Tasks to Perform Prior to the
Transformation Laboratory on page 34 for details.
Ligation Reaction
The pJet1.2 blunted vector is supplied ready to use, already opened, with
blunt ends ready for ligation to PCR products. The pJet1.2 plasmid selects
successful ligations through the disruption of an otherwise lethal gene,
eco47IR, which enables positive selection of the recombinants. Before
ligation, a 3'-A overhang must be removed from the PCR products by
treating the PCR product with a proofreading DNA polymerase, leaving
them with blunt ends ready to be ligated to the pJet1.2 blunted vector. This
DNA polymerase is active at 70°C but not at lower temperatures, so it is
not necessary to inactivate this enzyme after use. Once blunted, the PCR
product is combined with the plasmid and T4 DNA ligase under conditions
optimal for ligation. The ligation reaction is fast, complete in 5–10 min. Only
a minimal increase in the number of transformants is gained by extending
the ligation time beyond 10 min. Following ligation of a gene into a plasmid,
students will perform transformation to introduce the plasmid into competent
E. coli cells.
Note: Prior to starting this laboratory activity, students must have already
performed a polymerase chain reaction (PCR) to amplify the DNA fragment
they wish to clone and subsequently purify the PCR product to remove
excess primers, nucleotides, and DNA polymerase, which would otherwise
interfere with subsequent experiments.
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