1 / 13

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

onan
Download Presentation

Isolation and Purification of DNA from Escherichia coli

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related