MB206 Microbial Biotechnology
Group:
EXPERIMENT 2: Extraction of Human Genomic DNA from buccal cells.
Introduction:
The Wizard ® Genomic DNA Purification Kit is designed for isolation of DNA from white
blood cells, tissue culture cells and animal tissue, plant tissue, yeast and Gram positive and
Gram negative bacteria. Generally, all methods involve the disruption and lysis of cells. This
is followed sometimes by the removal of RNA (by RNAses, salt or other methods). Choosing
which method to use will depend on many selection factors including:
DNA is isolated from proteins by several methods including digestion of proteins by the
enzyme proteinase K. Proteins are removed subsequently by salting-out, organic extraction,
or binding of the DNA to a solid-phase support (such as an anion-exchange column or silica
technology). DNA is finally recovered by ethanol precipitation or isopropanol precipitation.
In general, the separation of DNA from cells and cellular components can be divided into
four stages:
1. Cell disruption
2. Lysis of Cell
3. Removal of Proteins and Contaminants
4. Recovery of DNA
In some genomic DNA isolation protocols, stages 1 and 2 are combined.
The Wizard ® Genomic DNA Purification Kit is based on a four-step process.
1. Lyses the cells and the nucleic.
2. Digest RNA using RNase.
3. Remove cellular proteins by a salt precipitation step, which precipitates the proteins
but leaves the high molecular weight genomic DNA in solution.
4. Finally, the genomic DNA is concentrated and desalted by isopropanol precipitation.
Objectives:
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MB206 Microbial Biotechnology
Methods and material:
Results
A260:
A280:
Ratio A260/280:
DNA concentration:
Comment on the steps, ratio and DNA concentration: (any contamination)
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MB206 Microbial Biotechnology
Question:
1. What is genomic DNA?
2. How is the DNA being separated from the other cellular components?
3. What role does centrifugation play?
4. What role does the NaCl solution play and NaCl added in which buffer? Please explain.
5. What is the role of the ethanol?
6. How to check the quality of your isolated genomic DNA?
Conclusion:
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MB206 Microbial Biotechnology
Suggestion (if any)
References (if any)
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