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

Why E. coli? A representative of prokaryotes A representative of prokaryotes E. coli is easy to culture in the laboratory. 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. 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. DNA is similar to all; serves as a model for understanding properties of human DNA.

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.

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 min. Cool to room temp. for 5 min.

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).

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.

Pour off the Ethanol and drain the tube on clean absorbent paper. Air dry the pellet for 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.

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.

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.

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

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References Isolaion and Purification of Total Genomic DNA from E. coli. A/genomic.pdf