Isolation of Nucleic Acids

Isolation of Nucleic Acids

Goals:

* removal of proteins
* DNA vs RNA
* isolate specific type of nucleic acid

Types of Methods:

* differential solubility
* ‘adsorption’ methods
* density gradient centrifugation

Types of DNA:

* genomic (chromosomal)
* organellar (satellite)
* plasmid (extra-chromosomal)
* phage/viral (ds or ss)
* complementary (mRNA)

General Features:

* denaturing cell lysis (SDS, alkali, heating, chaotropic)
*  enzyme treatments
o protease
o RNase (DNase-free)
o DNase (RNase-free)

High MW Genomic DNA Isolation

Typical Procedure

* Cell Lysis
o 0.5% SDS + proteinase K (55o several hours)
* Phenol Extraction
o gentle rocking several hours
* Ethanol Precipitation
* RNAse followed by proteinase K
* Repeat phenol extrac-tion and EtOH ppt

Phenol Extraction

* mix sample with equal volume of sat. phenol soln
* retain aqueous phase
* optional chloroform/isoamyl alcohol extraction(s)

 aqueous phase (nucleic acids)

 phenol phase (proteins)
and the the following steps can be found at the presentations

Isolation of Nucleic Acids.pps

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