# Nucleic Acid Purification Like DNA, **purification of RNA** is often the first steps for many experiments such as cDNA generation or [[Northern Blot]] analysis. Because RNA is less stable than DNA, its purification is often more challenging: > [!summary]- DNA vs RNA > See also: [[Nucleic Acids]] > ![[Nucleic Acids#DNA vs RNA]] ## RNA Purification **mRNA** (often the target) may be as low as *1-5%* of all RNA present within a cell. - The *polyA* tail of mRNA will bind to **oligo-dT** (repeated Ts) beads or columns, allowing affinity [[chromatography]] to be used for purification Even if general RNA is purified from a sample, mRNA specifically will have to be further purified from the sample later on. --- RNA Purification: 1. Solubilize Cellular Componenets 2. Denature Proteins 3. Remove Lipids 4. Inactivate RNases There are various RNA purification techniques: ### TRIzol > Developed by Chomczynski and Sacchi (1987), 5th most cited paper RNA purification via TRIzol uses phenol, chloroform, and guanidium isothiocyanate to denature proteins and inactivate intracellular RNases. A different pH is used for RNA and DNA separation steps, causing DNA to be in the organic phase, RNA to be in the aqueous phase, and proteins at the interphase. - pH = 4.8 for RNA - pH = 7.5 for DNA ![[RNA Purification and Quantification.png|600]] ## DNA Purification The **purification of genomic DNA** is often the *starting point* for further experiments - Ex: gene cloning, [[Polymerase Chain Reaction (PCR)|PCR]], [[DNA Hybridization]] The general protocal for purifying DNA involves: 1. [[Cell Disruption]] 2. Removal of Contaminants 3. DNA Purification on a Column (Miniprep) Unlike proteins, DNA can be purified using *organic solvents* and also *require small quantities* ### Preventing DNA Damage Many protocols use **Proteinase K** which degrades most proteins, especially nucleases (whose job is to destroy nucleic acids). - Can remain active in the presence of SDS, EDTA, urea, and within a wide pH range. ### Purification Techniques | Name | Description | | --- | --- | | Crude Lysate | Heat Cells & Add Proteinase K | | Salting Out/Precipitation | Performed using potassium acetate, centrifugation, and alcohol | | Anion Exchange | Backbone binds to positively charged beads, alcohol precipitation | | Cesium Chloride Gradient | High purity but very time-consuming | | Phenol-Chloroform Extraction | Phenol (aqueous, contains DNA and RNA), chloroform (organic, contains proteins and cellular debris), isoamyl alcohol. Is a tedius technique, can contain residual organics and hazardous waste, may inhibit downstream experiments like PCR. | | Alkaline Lysis | Using SDS to break up phospholipid bilayer and NaOH to dissolve structural proteins along with agitation, precipitation, and centrifugation | | Silica-based Methods | Selective absorption of DNA to silica-based gels in the presence of chaotropic salts | Most DNA purification kits (such as the Miniprep seen below) combine several of these techniques. ### DNA Preparation (Miniprep, Maxiprep) > See also: > - [[Recombinant DNA Technology]] Often times, cloned genes are **stored in plasmids** (as recombinant DNA), rather than as free DNA. After these recombinant plasmids are grown from a bacteria colony, they *must be purified before they can be used in further analyses*. **Miniprep** (minipreparation) is a kit-based technique that combines *alkaline lysis and silica-based beads* (Can yield 50-100μg of DNA). - Other variations occur that can yield much higher quantities, up to 15 mg (Midiprep, Maxiprep, Megaprep, Gigaprep)