Dna isolation
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:
- Biological methods such as using lytic enzymes.
- Physical methods such as using freeze-thaw and osmotic pressure to burst the cells.
- 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 preproducting 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
- ↑ Principles and techniques of biochemistry and molecular biology, 7th Edition Wilson K, Walker J Cambridge University Press, 2009
- ↑ K. Smith, M. A. Diggle, S. C. Clarke (2003)fckLRBacteriology Issue 41, Volume 6, Pages 2440-2443fckLRDOI:10.1128/JCM.41.6.2440-2443.2003fckLRhttps://jcm.asm.org/content/41/6/2440