Dna isolation: Difference between revisions
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Step 1- | Step 1- Isolate [[DNA|DNA]] out of the cell | ||
Step 2 - Centrifuge down the fragments at low rpm | 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 3 - Separate DNA from proteins and lipids by adding equal volume of [[Phenol|phenol]]/[[Chloroform|chloroform]] | ||
Step 4 - Concentrate DNA using 2 volumes of ethanol | Step 4 - Concentrate DNA using 2 volumes of ethanol | ||
Step 5 - Confirm DNA purity - A260/A280 > 1.8 indicates pure DNA | Step 5 - Confirm DNA purity - A260/A280 > 1.8 indicates pure DNA<ref>Principles and techniques of biochemistry and molecular biology, 7th Edition Wilson K, Walker J Cambridge University Press, 2009</ref>. | ||
DNA is extracted from cells by lysis. The different types of lysis include: | |||
#Biological methods such as using [[Lytic enzymes|lytic enzymes]]. | |||
#Physical methods such as using [[Freeze-thaw|freeze-thaw]] and [[Osmotic pressure|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 reproducing experiments<ref>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</ref>. 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 === | |||
<references /> | |||
Latest revision as of 17:25, 18 October 2018
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 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
- ↑ 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) 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