Bio-Rad Ligation and Transformation Module - Manual (Page 6)

Using the Ligation and Transformation module, students will clone a gene
of interest. Prior to starting this laboratory activity, students must have
already amplified a gene of interest using polymerase chain reaction
(PCR) and subsequently purified the PCR product to remove excess
primers, nucleotides, and DNA polymerase, which would otherwise interfere
with subsequent experiments. Students can then use the Ligation and
Transformation module to ligate the DNA fragment into the pJet1.2 blunted
vector, which encodes
amp r , an ampicillin-resistance gene. Following ligation,
students will perform transformation to introduce the plasmid into living
bacterial cells.
The pJet1.2 blunted vector enables positive selection of plasmids with the
desired insert due to the disruption of
eco47IR, an otherwise lethal gene,
that allows growth of successful transformants. Bacteria are then plated
and incubated overnight at 37°C on the selective medium containing
ampicillin and isopropyl
b-D-1-thiogalactopyranoside (IPTG), which is
added to increase expression of the ampr gene. Since transformed cells
express an ampicillin-resistance gene, they will grow and divide, each
forming a colony on the plate that is the product of a single transformation
event.
The bacteria containing the cloned gene can be grown in liquid growth
medium and the plasmid containing the insert can be purified from the
bacteria. The pJet1.2 plasmid contains a Bgl II restriction enzyme recognition
site on either side of the insertion site. Using the Bgl II enzyme students
will analyze the cloned plasmid by restriction enzyme digestion and analyze
their digests by agarose gel electrophoresis to confirm the presence of an
insert and determine its size. The resulting fragment can then be compared
to the size of the PCR fragment ligated into the plasmid. Finally, the DNA
fragment can be then sequenced to determine the exact order of
nucleotides in the DNA molecule.
What Skills Do Students Need to Perform this Laboratory
Activity?
This laboratory activity assumes that students and instructors have basic
molecular biology and microbiology skills, such as proper pipeting
techniques, pouring and streaking agar plates and performing agarose gel
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