DNA Extraction and Purification

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Extraction of [[DNA|DNA]] from a source organism (animal, plants or microbes) is easier when compared to using the same technique to isolate [[RNA|RNA]].This is because DNA is very stable and is the same in all cells of multicellular organism. The key steps in genomic DNA isolation are:
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Extraction of [[DNA|DNA]] from a source organism (animal, plants or microbes) is easier when compared to using the same technique to isolate [[RNA|RNA]].This is because DNA is very stable and is the same in all cells of multicellular organism. The key steps in genomic DNA isolation are:  
  
 
1) Disruption of cell membranes(animal cells)/cell walls(plant cells)  
 
1) Disruption of cell membranes(animal cells)/cell walls(plant cells)  
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B) Chemical Approaches   
 
B) Chemical Approaches   
  
This is the use of [[Enzymes|enzymes]] such as [[Lysozyme|lysozyme]] and detergents on cell components or cytosolic domains to cause lysis of the cells. [[Lysozyme|Lysozyme]] is often added to detergents in this process because of its ability to digest cell wall components of [[gram-positive|gram-positive]] bacteria  
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This is the use of [[Enzymes|enzymes]] such as [[Lysozyme|lysozyme]] and detergents on cell components or cytosolic domains to cause lysis of the cells. [[Lysozyme|Lysozyme]] is often added to detergents in this process because of its ability to digest cell wall components of [[Gram-positive|gram-positive]] bacteria  
  
 
2) Low speed centrifugation of isolated cells to remove debris  
 
2) Low speed centrifugation of isolated cells to remove debris  
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3) Addition of [[RIBONUCLEASE|Ribonuclease]] to destroy RNA  
 
3) Addition of [[RIBONUCLEASE|Ribonuclease]] to destroy RNA  
  
4) Removal of contaminants by mixing with equal volumes of [[phenol/chloroform|phenol/chloroform]]. This denatures proteins and they are precipitated to form an interface layer in the test tube.  
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4) Removal of contaminants by mixing with equal volumes of [[Phenol/chloroform|phenol/chloroform]]. This denatures proteins and they are precipitated to form an interface layer in the test tube.  
  
 
5) Concentration of DNA by precipitation  
 
5) Concentration of DNA by precipitation  
  
This is done by adding 2 volumes of [[ethanol|ethanol]] plus 0.2M NaCl.  
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This is done by adding 2 volumes of [[Ethanol|ethanol]] plus 0.2M NaCl.  
  
6) Measurement of DNA concentration<br>  
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6) Measurement of DNA concentration<br>
  
This can be done by using either of these two methods;  
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This can be done by using&nbsp;either of these two methods;  
  
 
i) Absorbance method&nbsp;  
 
i) Absorbance method&nbsp;  
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ii) Flourescence method  
 
ii) Flourescence method  
  
However,the simplest is the absorbance method which is carried using [[Spectrophotometry|Spectrophotometry]]. This uses a simple laboratory equipment called Spectrophotometer including UV lamp, transparent curvettes and a solution of purified DNA. Absorbance readings is taken at 260nm,this is because DNA absorbs light more at this [[wavelength|wavelength]].
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However,the simplest is the absorbance method which is carried using [[Spectrophotometry|Spectrophotometry]]. This uses a simple laboratory equipment called Spectrophotometer including UV lamp, transparent curvettes and a solution of purified DNA. Absorbance readings is taken at 260nm,this is because DNA absorbs light more at this [[Wavelength|wavelength]].

Revision as of 14:43, 17 October 2013

Extraction of DNA from a source organism (animal, plants or microbes) is easier when compared to using the same technique to isolate RNA.This is because DNA is very stable and is the same in all cells of multicellular organism. The key steps in genomic DNA isolation are:

1) Disruption of cell membranes(animal cells)/cell walls(plant cells)

This can be done using two approaches;

A) Physical Approaches

i) Sonication: the process of directing vibration generated from a mechanical process towards a substance(e.g DNA). Bacteria cells containing DNA are subjected to sonication to break the cell membranes and release the DNA proteins. An equipment known as a Sonicator is used for this process.

ii) Shearing: this is the accidental tearing apart of long DNA molecules during laboratory preparations. It may also be done intentionally.

B) Chemical Approaches 

This is the use of enzymes such as lysozyme and detergents on cell components or cytosolic domains to cause lysis of the cells. Lysozyme is often added to detergents in this process because of its ability to digest cell wall components of gram-positive bacteria

2) Low speed centrifugation of isolated cells to remove debris

3) Addition of Ribonuclease to destroy RNA

4) Removal of contaminants by mixing with equal volumes of phenol/chloroform. This denatures proteins and they are precipitated to form an interface layer in the test tube.

5) Concentration of DNA by precipitation

This is done by adding 2 volumes of ethanol plus 0.2M NaCl.

6) Measurement of DNA concentration

This can be done by using either of these two methods;

i) Absorbance method 

ii) Flourescence method

However,the simplest is the absorbance method which is carried using Spectrophotometry. This uses a simple laboratory equipment called Spectrophotometer including UV lamp, transparent curvettes and a solution of purified DNA. Absorbance readings is taken at 260nm,this is because DNA absorbs light more at this wavelength.

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