Bio-Rad Ligation and Transformation Module - Manual (Page 37)

Note: Components needed to carry out PCR reactions are not included in
the Ligation and Transformation module. A kit that utilizes size exclusion
chromatography, such as Bio-Rad’s PCR Kleen™ Spin Purification module
(catalog #732-6300EDU), can be used for purifying the PCR products.
Once the purified PCR product is available, students may decide to
electrophorese the PCR products to assess the quality and quantity of the
sample. Electrophoresis of the purified sample is an optional activity and
could be skipped.
Tasks to Perform Prior to the Ligation Laboratory
1. Thaw the 2x ligation reaction buffer, proofreading polymerase, T4 DNA
ligase, pJet1.2 blunted vector, purified PCR fragment, and sterile water
and store on ice. Just before use, mix and centrifuge the reagents to
collect contents at the bottom of the tubes. Do not aliquot the ligation
reagents for students; the volumes required are too small. Have these
reagents available at the common workstation for students. Also ensure
students are familiar with methods to pipet small volumes.
Transformation Laboratory
At this transformation step, students will transform bacteria with the liga-
tion reaction. Following transformation, pJet1.2 enables positive selection
of plasmids with the desired insert due to the disruption of an otherwise
lethal gene,
eco47IR, which allows growth of successful transformants.
Bacterial transformation with ligation reactions is a very inefficient process
(much more inefficient than transformation with plasmid DNA), so students
should be encouraged to take special care during this protocol. There are
many steps that if performed improperly can lead to reduced transformation
efficiency or even no colonies. It is recommended that students perform a
control transformation using 1 µl of 50–200 ng/µl of an ampicillin resistant
control plasmid if available. (pGLO Plasmid (20 µg) (catalog #166-0405EDU)
can be purchased separately and used as a control.) Each student team
would require an additional LB Amp IPTG agar plate for their control
transformation. Note: Each preparation of competent cells derives sufficient
cells for two transformations.
The transformation protocol involves creating competent cells and
immediately performing the transformation. Once made, the competent
cells must be used immediately or discarded according to local regulations.
They cannot be stored for later use. This transformation method permits
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