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Isolation and Purification of DNA from Escherichia coli

Isolation and Purification of DNA from Escherichia coli. GROUP 2 Chester Mancia Frances Miclat Mark Mosses Oliva HUB 42. DNA isolation. is a routine procedure to collect DNA for subsequent molecular analysis. Four Basic Steps: Cell disruption by a lysis solution

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Isolation and Purification of DNA from Escherichia coli

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  1. Isolation and Purification of DNA from Escherichia coli GROUP 2 Chester Mancia Frances Miclat Mark Mosses Oliva HUB 42

  2. DNA isolation • is a routine procedure to collect DNA for subsequent molecular analysis. • Four Basic Steps: • Cell disruption by a lysis solution • Removing membrane lipids by a detergent • Removing proteins by a protease • Precipitating the DNA with an alcohol

  3. Why E. coli? A representative of prokaryotes E. coli is easy to culture in the laboratory. It is easier to extract DNA from a bacterium because the DNA is not enclosed in a nuclear membrane. DNA is similar to all; serves as a model for understanding properties of human DNA.

  4. Procedure: Transfer E. coli to microcentrifuge tube. Centrifuge at 5,000 rpm for 5 min. Discard supernatant and collect cell pellet Resuspend in 600µl Lysis Solution Incubate at 80°C for 5 min.

  5. Pipet until cells are thoroughly suspended. Cool to room temperature for 5 min. Add 3µl of RNase solution and invert the tube 2-5 times. Incubate at 37°C for 15-60 min. Cool to room temp. for 5 min.

  6. Add Protein Precipitation Solution(PPS). Vortex at high speed for 20 seconds. Incubate in Ice for 5 min. Centrifuge at 15,000 rpm for 3 min. Transfer the supernatant to a microcentrifuge tube w/ 600 µl Isopropanol(IPA).

  7. Gently mix by inversion. Centrifuge at 15,000 rpm for 3 min. Pour off the supernatant and drain the tube on clean absorbent paper(Do not dry out the pellet) Add 600µl 70% ethanol and invert the tube several times to wash the DNA pellet. Centrifuge at 15,000 rpm for 3 min.

  8. Pour off the Ethanol and drain the tube on clean absorbent paper. Air dry the pellet for 10-15 min. Add 100µl DNA Rehydration Solution(RH). Incubate at 65°C for 1 hr. to rehydrate the DNA. Gently tap the tube to mix and store the DNA at 2-8°C.

  9. Guide Questions • What is the rationale of adding Cell Lysis Solution? • Also called SDS (Sodium Dodecyl Sulfate) • Lysis Solution is an anionic detergent that breaks apart the lipid membrane and solubilizes cellular proteins.

  10. Addition of Protein Precipitation Solution • To precipitate the protein components of the lysed cell of E. coli. • Proteins are subjected to chemical denaturation and/or enzymatic degradation to exploit the DNA of the cell. • The solution take advantage of different molecular weights of the cellular components to precipitate the protein without damaging the DNA.

  11. Addition of Hydration Solution • to be able to hydrolyze the DNA • Subsequent procedure (addition of ethanol) degrades the DNA to loose its Hydrogen component on the minor groove. • Hydration gives the H+ back to the DNA

  12. END! Thank You for Listening!

  13. References • Isolaion and Purification of Total Genomic DNA from E. coli. http://bio.classes.ucsc.edu/bio105l/EXERCISES/DNA/genomic.pdf

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