Bio-Rad Ligation and Transformation Module - Manual (Page 5)

Introduction
Cloning is the production of multiple exact copies of a piece of DNA, usually
a gene, using molecular biology techniques. Cloning is frequently the first
step of a research project, producing enough DNA for further study.
Using the Ligation and Transformation module students can subclone virtually
any DNA fragment of interest that has been amplified using PCR. We
recommend that the DNA fragment be approximately 200–2,000 base pairs
(bp) in length for best results. Below is a typical workflow for cloning and
sequencing a gene. The steps that the Ligation and Transformation module
enable students to perform are in bold.
The Ligation and Transformation module is part of Bio-Rad’s Cloning and
Sequencing Explorer Series. The Cloning and Sequencing Explorer Series
is a sequence of individual modules that have been designed to work in
concert to give students the real world experience of a molecular biology
research workflow. The additional modules of the Cloning and Sequencing
Explorer Series can be purchased separately. Further information on the
separate modules is available in the Biotechnology Explorer™ catalog or
from explorer.bio-rad.com.
• Amplify gene of interest using PCR 1
• Purify PCR product 2
• Ligation of PCR product into pJet1.2 plasmid
• Transform ligated plasmid into bacteria
• Culture bacteria and grow minipreps 3
• Purify plasmid from minipreps 4
• Analyze plasmid by restriction digestion
• Electrophorese restriction digest reaction 5
• Sequence plasmid and analyze 6
1
GAPDH PCR module (catalog #166-5010EDU) amplifies a fragment of the GAPDH gene
from a preparation of plant genomic DNA.
2 PCR Kleen™ Spin module (catalog #732-6300EDU) purifies 25 PCR products.
3 Microbial Culturing module (catalog #166-5020EDU) contains all required reagents for
culturing bacteria for transformation using the Ligation and Transformation module.
4 Aurum™ Plasmid Mini Purification module (catalog #732-6400EDU) contains reagents to
purify plasmid DNA from 100 minipreps.
5 Electrophoresis modules contain reagents to analyze plasmid restriction digests.
6 Sequencing and Bioinformatics module (catalog #166-5025EDU) is designed to allow
sequencing and bioinformatics analysis of plasmids generated using the Ligation and
Transformation module.
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