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Team:Cambridge/Protocols/Mini Prep
From 2011.igem.org
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===Theory=== | ===Theory=== | ||
| - | + | The miniprep kit relies on a column, which fits into a centrifuge tube which has different affinity for DNA depending on buffer which is washing through. | |
| + | First centrifugation causes the cells to form a pellet at the bottom of the tube, this allows the medium in which the bacteria have been growing to be removed (the supernatant) and the cells can then be resuspended in another chemical. This prinicple is used in many protocols to allow the solution surrounding the cell replaced with various buffers in the kit. | ||
| + | Bacterial cells are lysed by exposing them to a detergent (SDS) which solubilises the cell membrane, whilst sodium hydroxide disrupts the cell wall and more importnantly denatures the genomic DNA, the plasmid DNA and the proteins. | ||
| + | The lysate is neutralized and adjusted to high salt concentration which causes denatured proteins, chromosomal DNA, cellular debris, and SDS to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution. | ||
| + | The chromosomal DNA is separated from the plasmid DNA because the chromsomal DNA binds to the QIAGEN membrane in the column along with the precipitating proteins and debris, whilst the plasmid DNA stays in solution and can be removed in the supernatant. | ||
| + | |||
===Practice=== | ===Practice=== | ||
====Preparation==== | ====Preparation==== | ||
Revision as of 14:38, 14 July 2011
Protocol Name
protocol description
Theory
The miniprep kit relies on a column, which fits into a centrifuge tube which has different affinity for DNA depending on buffer which is washing through. First centrifugation causes the cells to form a pellet at the bottom of the tube, this allows the medium in which the bacteria have been growing to be removed (the supernatant) and the cells can then be resuspended in another chemical. This prinicple is used in many protocols to allow the solution surrounding the cell replaced with various buffers in the kit. Bacterial cells are lysed by exposing them to a detergent (SDS) which solubilises the cell membrane, whilst sodium hydroxide disrupts the cell wall and more importnantly denatures the genomic DNA, the plasmid DNA and the proteins. The lysate is neutralized and adjusted to high salt concentration which causes denatured proteins, chromosomal DNA, cellular debris, and SDS to precipitate, while the smaller plasmid DNA renatures correctly and stays in solution. The chromosomal DNA is separated from the plasmid DNA because the chromsomal DNA binds to the QIAGEN membrane in the column along with the precipitating proteins and debris, whilst the plasmid DNA stays in solution and can be removed in the supernatant.
Practice
Preparation
Safety
The safety implication of the procedure.
