Dna isolation

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Step 1- Isolate DNA out of the cell

Step 2 - Centrifuge down the fragments at low rpm

Step 3 - Separate DNA from proteins and lipids by adding equal volume of phenol/chloroform

Step 4 - Concentrate DNA using 2 volumes of ethanol

Step 5 - Confirm DNA purity - A260/A280 > 1.8 indicates pure DNA[1].

DNA is extracted from cells by lysis. The different types of lysis include:

  1. Biological methods such as using lytic enzymes.
  2. Physical methods such as using freeze-thaw and osmotic pressure to burst the cells.
  3. Mechanical methods such as grinding or shearing.

Grinding methods include using a pestle and mortar, primarily used for plant cells, using a bead mill for tough samples or vortexing.

Shearing methods include using a homogeniser, a rotor-stator or syringe needle.

DNA may be purified by adding phenol-chloroform mixture or via a commercial kit which is a lot easier and quicker. Commercial methods are preferred due to accuracy and ease of reproducing experiments[2]. The DNA retrieved via these kits is also usually purer than when Phenol-Chloroform is used. The kits are also not hazardous, unlike the Phenol-Chloroform version.

References

  1. Principles and techniques of biochemistry and molecular biology, 7th Edition Wilson K, Walker J Cambridge University Press, 2009
  2. K. Smith, M. A. Diggle, S. C. Clarke (2003) Bacteriology Issue 41, Volume 6, Pages 2440-2443 DOI:10.1128/JCM.41.6.2440-2443.2003 https://jcm.asm.org/content/41/6/2440
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