plasmid
Experiment 22
Laboratory to Biology III Diversity of Microorganisms / Wintersemester / page 1
Experiment 22
Isolation of plasmid-DNA from bacteria and PCR
Advisor
Konrad Egli: kegli@botinst.unizh.ch
Reading
Chapters in BBOM 10th: 10.8
BBOM : Madigan M.T., J.M. Martinko and J. Parker: "Brock - Biology of
Microorganisms", 10th Edition (2003), Prentice Hall.
Objectives
Background
• Isolation of plasmid-DNA from different bacteria clones
• Handling of bacteria clones
• PCR-experiment
The typical plasmid is a circular double-standed DNA molecule less than 1/20 the size of the chromosome. Most plasmids are circular, but linear plasmids are also known. Naturally occuring plasmids vary in size from 1 to more than 1000 kilobase pairs. Most of the plasmid-DNA isolated from cells is in the supercoiled configuration, which is the most compacted form within the cell.
Isolation of plasmid-DNA can generally be accomplished by making use of the physical properties of supercoiled DNA molecules. Although chromosomes are also supercoiled inside the cell, isolation of chromosomal DNA almost always leads to breakage of the strands and consequent loss of supercoiling. Separation can then proceed by a variety of techniques, including ultracentrifugation and electrophoresis on agarose gels.
Among the most widespread and well-studied groups of plasmids are the resistance plasmids which confer resistance to antibiotics and various other inhibitors of growth.
Today, there are are number of well established techniques commercially available as kits which make DNA extraction and purification easy. Although kits offer fast and reliable methods for extracting plasmid DNA with high yields, kits are expensive. Since a PCR is performed afterwards, which amplifies certain DNA fragments, we use miniaturized extraction methods; the extracted amount is large enough for further processing.
PCR, a method to amplify DNA: For details see lectures in
Laboratory to Biology III Diversity of Microorganisms / Wintersemester / page 1
Experiment 22
Isolation of plasmid-DNA from bacteria and PCR
Advisor
Konrad Egli: kegli@botinst.unizh.ch
Reading
Chapters in BBOM 10th: 10.8
BBOM : Madigan M.T., J.M. Martinko and J. Parker: "Brock - Biology of
Microorganisms", 10th Edition (2003), Prentice Hall.
Objectives
Background
• Isolation of plasmid-DNA from different bacteria clones
• Handling of bacteria clones
• PCR-experiment
The typical plasmid is a circular double-standed DNA molecule less than 1/20 the size of the chromosome. Most plasmids are circular, but linear plasmids are also known. Naturally occuring plasmids vary in size from 1 to more than 1000 kilobase pairs. Most of the plasmid-DNA isolated from cells is in the supercoiled configuration, which is the most compacted form within the cell.
Isolation of plasmid-DNA can generally be accomplished by making use of the physical properties of supercoiled DNA molecules. Although chromosomes are also supercoiled inside the cell, isolation of chromosomal DNA almost always leads to breakage of the strands and consequent loss of supercoiling. Separation can then proceed by a variety of techniques, including ultracentrifugation and electrophoresis on agarose gels.
Among the most widespread and well-studied groups of plasmids are the resistance plasmids which confer resistance to antibiotics and various other inhibitors of growth.
Today, there are are number of well established techniques commercially available as kits which make DNA extraction and purification easy. Although kits offer fast and reliable methods for extracting plasmid DNA with high yields, kits are expensive. Since a PCR is performed afterwards, which amplifies certain DNA fragments, we use miniaturized extraction methods; the extracted amount is large enough for further processing.
PCR, a method to amplify DNA: For details see lectures in