Download as PDF, PPTX
Uploaded byNetHelix
Spin column extraction method molecular biology course.pdf
The document discusses the advantages of spin column extraction methods for nucleic acid, highlighting efficiency, scalability, and contamination prevention. It details the mechanisms of various types of spin columns, including silica membrane, anion exchange, and filter paper-based columns, focusing on their binding properties and operational processes. The elution methods for DNA and RNA are also provided, emphasizing conditions for optimal stability and solubility.
More Related Content
Similar to Spin column extraction method molecular biology course.pdf
Spin column extraction method molecular biology course.pdf
- 1.
- 2. Advantages less time consumption Economical energy- efficient automated operation prevention from cross- contamination highyield environment friendly adaptability significant scalability
- 3. commercial types ofspin column used for nucleic acid extraction
- 4. Silica membrane • Themost popular and widely used technique • The spin column contains a silica membrane as the gel material • DNA or RNA binds to silica by adsorption • Role of Chaotropic Agents: • Denaturation and Hydrophobicity Reduction: Chaotropic agents like guanidinium HCl denature biomolecules and reduce their hydrophobicity. • Salt Bridge Formation: In the presence of high salt concentrations, chaotropic agents facilitate the formation of a positive salt bridge between the negatively charged silica and the negatively charged nucleic acid molecules. • Avoiding Contamination and Shearing: The silica is bound to a solid support in the spin column, which helps avoid issues such as glass-bead contamination of extracted nucleic
- 5. Anioin exchange Anionexchange- based spin column • Used in most transfection-grade plasmid DNA extraction and purification • positively charged resin (like diethylaminoethyl cellulose or DEAE cellulose): attracts and binds the negatively charged phosphate groups of the nucleic acid molecules.
- 6. Filter paper-based spincolumn • similar to silica membrane-based principle where isolation in the presence of chaotropic agents. • The secondary fibril-associated cellulose of filter paper binds to the nucleic acids in chaotropic conditions • Filter paper shows weak binding affinity to the plasmid DNA.
- 7.
- 8. 5 – stagesprocess
- 9. Cell lysis Lytic buffer Chaotropicsalts Detergent Enzymes Urea guanidine thiocyanate Proteinase K Lysozyme SDS
- 10. purification Ethanol / Isopropanol Sodium Chloride (NaCl) Shouldbe in optimal amount lower amount impedes the washing of the salt from the membrane. A high amount of ethanol can cause degradation
- 11.
- 12. Dry spin • Purposeof this stage: • Remove Residual Ethanol • Prepare for Elution
- 13. Elution For DNA • DNAis stable in a basic pH medium and dissolves in it faster than water. • A basic buffer of 10 mM Tris at pH 8–9 is generally used. For RNA • water is used as eluent (RNase-free Water)as water pH is 4–5. This is because nucleic acid is more stable and dissolves easily in slightly acidic pH.
- 19. References • Gautam, A.(2022). Spin column-based isolation of nucleic acid. In DNA and RNA Isolation Techniques for Non-Experts (pp. 47-53). Cham: Springer International Publishing. • Shin, J. H. (2013). Nucleic acid extraction techniques. Advanced Techniques in Diagnostic Microbiology, 209-225 • Shin J. H. (2012). Nucleic Acid Extraction Techniques. Advanced Techniques in Diagnostic Microbiology, 209–225. https://doi.org/10.1007/978-1-4614-3970-7_11 • Shi, R., Lewis, R. S., & Panthee, D. R. (2018). Filter paper-based spin column method for cost-efficient DNA or RNA purification. PloS one, 13(12), e0203011. https://doi.org/10.1371/journal.pone.0203011