DNA isolation molecular biology practical.pptx

molecular biology

1. Nucleic acid Isolation Nucle ic acid Isolation

2. DNA isolation : * aims to collect as much DNA DNA purification : * Reduce contamination from isolated DNA DNA extraction : * achieve both isolation and purification

3. Is a method to purify DNA by using physical and / or chemical methods from a sample Separating DNA from cell membrane , proteins, and other cellular components. What is extraction

4.   Molecular studies , diagnosis , forensic science , vaccine development , pharmaceuticals. Sample source : microorganism , human , plant sample type : Blood ,urine ,biopsy ,sputum , hair , bones ,semen Nucleic acid type ; RNA , DNA Planned usage : PCR , sequencing ,fingerprinting Importance and application of DNA , RNA extraction choosing best extraction method depend on ;

5.   DNA. Found within cell Eukaryotic : inside nucleus Prokaryotic : inside cytoplasm Inside cells with enzymes ,protein ,organic and in organic compound all-non nucleic acid component need to be removed

6. Steps in DNA extraction : 1.lysis 2.precipitation 3.purification

7. Physical method Mechanical disruption plant cells, tough cell wall blender Mortar And pestle Cutting sonicator 1.Cell lysis /cell disruption Chemical method Detergents : Sodium Dodecyl Sulfate (SDS)/lauryl Sulfate (rapid disruption Enzymes : Target aminoacids bonds ProteinaseK;animal cell Cellulase;plant cell lyticase : yeast Lysozyme:gram positive bacteria

8. 2. Precipitation Separate DNA from cellular debris after lysis : cell extract :DNA mixed with cell debris and contaminants ( proteins, detergents,reagents,etc ) Sodium (Na+)ions. Neutralize negative charge More stable Alcohol : isopropanol/ethanol cause precipitation DNA insoluble with alcohol Protease : degrade DNA associated proteins Filtration

9. 3. Purification After precipitation ; DNA separated from aqueous phase Isopropanol or absolute ethanol Removal of cellular debris and unwanted material Storage and handling

10. Extraction methods : chemical DNA extraction method Physical DNA extraction method Organic DNA extraction method Inorganic DNA extraction method   Magnetic bead. DNA Extraction Paper DNA extraction method Phenol-chloroform DNA Extraction method Salting out method Silica gel based DNA extraction method Proteinase k DNA extraction method

11. Organic DNA extraction Plant sample

12. 1.Lysis : Extraction buffer Detergent : disruption of cell membrane   Reducing agent :protein denaturation Chelating agent :removal of Mg++ions Buffer Salt Sodium dodecyl sulfate(SDS ) Cetyl Trimethyl ammonium bromide(CTAB) B mercaptoethanol EDTA T ris : pH8 Nacl

13. Step 1 Cell Lysis Disruption of cell wall   Esp for plant E.g : blender , mortar and pestle ,liquid nitrogen Protein digestion Transfer ground plant samples into eppendorf tube Extraction buffer Lyse cell membrane and inhibit nuclease activity

14. . incubate at 60deg celsius for 20-40 mins in a water bath centrifugatio n 10000rpm,10mins removal of cell debris transf er supernatant (crude lysate ) to a new tube

15. 2.Precipitation Crude lysate: with nucleic acids and other cell constituents add equal amount of phenol -chloroform to the crude lysate *Mix by vortex centrifugation 10.000rpm 10mins separates aqueous phase , interphase and organic phase collect aqueous phase (with nucleic acids ) into new tube Do not disturb the organic phase Use wide bore tips :avoid mechanical DNA damage add ice cold 95%ethanol and salt to aqueous phase Mix by inversion -20 deg celsius 1hour - centrifugation 10.000rpm. 10min

16. 3.Purification   Decant supernatant * wash DNA pellet with 70% ethanol * mix by inversion * centrifugation 10.000 rpm5mins * Discard ethanol ( noresidual ethanol ) * *Dry pellet at RT for 30 mins *Dissolve and resuspend DNA pellet : TE buffer / nuclease free water DNA pellet

17. In organic DNA extraction (proteinase k method) Blood sample

18. Lysis ,Add 2ml blood with 10-20UL TE buffer centrifugation at 2500rpm for 20mins add 10-15UL TE buffer to pellet incubate at high temperature lyse cell and nuclear membrane Digest protein discard supernatant Mix gently 60-65 deg celsius 2hours

19. centrifugation 2500rpm for 15mins Add proteinase k solution and DNA extraction buffer incubate at high temperature Proteinase k discard supernatant . 60-65 deg celsius 2hours 60 deg c(37-70deg ) PH 8.0

20. Precipitation Add 1-2 ml chilled isopropanol and a pinch of Nacl mix by invertion Centrifugation at 8000-10000rpm discard supernatant

21. purification   Add 1ml ethanol to pellet centrifugation 10000 rpm for 1-2mins Add TE buffer and incubate at water bath for 2-3 hours Discard ethanol Dry pellet