DNA Isolation

Cheatsheet Content

### Genomic DNA Isolation Genomic DNA (gDNA) isolation typically involves cell lysis, removal of proteins and RNA, and DNA precipitation. #### 1. Cell Lysis - **Mechanical Lysis:** Grinding (e.g., with mortar and pestle for plant tissue), bead beating. - **Chemical Lysis:** - **Detergents:** SDS (sodium dodecyl sulfate) to disrupt cell membranes and denature proteins. - **Alkaline Lysis:** NaOH for bacterial cells, denatures DNA. - **Enzymatic Lysis:** - **Lysozyme:** For bacterial cell walls (peptidoglycan). - **Proteinase K:** Digests proteins, including nucleases. - **Cellulase/Pectinase:** For plant cell walls. #### 2. Protein and RNA Removal - **Protein Removal:** - **Proteinase K:** Common for digesting proteins. - **Salting Out:** High salt concentrations (e.g., ammonium acetate, potassium acetate) precipitate proteins. - **Phenol-Chloroform Extraction:** Organic solvents separate proteins and lipids from nucleic acids. (Hazardous, less common now). - **RNA Removal:** - **RNase A:** Digests RNA. #### 3. DNA Precipitation - **Cold Ethanol/Isopropanol:** DNA is insoluble in alcohol and precipitates out, especially in the presence of high salt. - **Salts:** Sodium acetate, ammonium acetate to neutralize the negative charge of DNA, aiding precipitation. - **Centrifugation:** To pellet the precipitated DNA. - **Washing:** 70% Ethanol wash to remove residual salts and contaminants. - **Resuspension:** In TE buffer (Tris-EDTA) or sterile water; EDTA chelates metal ions, inhibiting DNases. ### Plasmid DNA Isolation (Miniprep) Plasmid DNA isolation, often called miniprep, is a rapid method to extract small amounts of high-quality plasmid DNA from bacterial cultures. #### 1. Bacterial Culture & Harvesting - Grow bacterial culture containing plasmid overnight. - Centrifuge to pellet bacterial cells. #### 2. Cell Lysis (Alkaline Lysis Method) - **Resuspension:** Cells resuspended in Buffer P1 (Glucose, Tris-HCl, EDTA). EDTA chelates Mg2+, destabilizing cell membrane. - **Lysis:** Add Buffer P2 (NaOH, SDS). - **SDS:** Disrupts cell membranes, denatures proteins. - **NaOH:** Denatures chromosomal DNA and plasmid DNA. The small, supercoiled plasmid DNA can re-anneal later, while the large, linear chromosomal DNA cannot. - **Neutralization:** Add Buffer P3 (Potassium Acetate, Glacial Acetic Acid). - **Potassium Acetate:** Forms a precipitate with SDS and denatured proteins (including genomic DNA). - **Acetic Acid:** Neutralizes the solution, allowing plasmid DNA to re-anneal. - **Centrifugation:** To pellet the protein/gDNA/SDS precipitate. Plasmid DNA remains in the supernatant. #### 3. DNA Purification - **Spin Column Method:** - Supernatant loaded onto a silica-based spin column. - Plasmid DNA binds to silica in high salt conditions. - **Wash Buffers:** Wash with buffers containing ethanol to remove contaminants (proteins, salts). - **Elution:** Elute plasmid DNA with low-salt buffer (e.g., TE buffer or sterile water). #### 4. Alternative Purification (Phenol-Chloroform/Ethanol Precipitation) - **Phenol-Chloroform Extraction:** (If not using spin column) Removes remaining proteins and lipids. - **Ethanol Precipitation:** Precipitates plasmid DNA from the aqueous phase. - **Wash and Resuspend:** As with gDNA. ### Quality Assessment - **Spectrophotometry (A260/A280 ratio):** - **A260:** DNA concentration. - **A280:** Protein contamination. - **Ratio ~1.8:** Pure DNA. Lower indicates protein contamination. - **Spectrophotometry (A260/A230 ratio):** - **A230:** Contamination by salts, carbohydrates, phenol. - **Ratio ~2.0-2.2:** Pure DNA. Lower indicates contamination. - **Agarose Gel Electrophoresis:** - **Integrity:** Undegraded DNA appears as single, high molecular weight band (gDNA) or distinct supercoiled/nicked forms (plasmid). - **Purity:** Absence of RNA smear or protein bands. - **Concentration:** Comparison to DNA ladder. - **PCR/qPCR:** Functional assessment of DNA for downstream applications.