Isolation and Isolation and Purification of DNA Purification of DNA

from Escherichia colifrom Escherichia coli

GROUP 2 GROUP 2 Chester ManciaChester Mancia

Frances MiclatFrances Miclat

Mark Mosses OlivaMark Mosses Oliva

HUB 42HUB 42

DNA isolationDNA isolation is a routine procedure to collect is a routine procedure to collect

DNA for subsequent molecular DNA for subsequent molecular analysis.analysis.

Four Basic Steps:Four Basic Steps: Cell disruption by a lysis solutionCell disruption by a lysis solution Removing membrane lipids by a Removing membrane lipids by a

detergentdetergent Removing proteins by a proteaseRemoving proteins by a protease Precipitating the DNA with an Precipitating the DNA with an

alcoholalcohol

Why E. coli?Why E. coli?

A representative of prokaryotesA 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 It is easier to extract DNA from a

bacterium because the DNA is not bacterium because the DNA is not enclosed in a nuclear membrane.enclosed in a nuclear membrane.

DNA is similar to all; serves as a model DNA is similar to all; serves as a model for understanding properties of human for understanding properties of human DNA.DNA.

Procedure:Procedure:Transfer E. coli to microcentrifuge Transfer E. coli to microcentrifuge

tube.tube.

Centrifuge at 5,000 rpm for 5 min.Centrifuge at 5,000 rpm for 5 min.

Discard supernatant and collect cell Discard supernatant and collect cell pelletpellet

Resuspend in 600µl Resuspend in 600µl Lysis SolutionLysis Solution

Incubate at 80°C for 5 min.Incubate at 80°C for 5 min.

Pipet until cells are thoroughly Pipet until cells are thoroughly suspended.suspended.

Cool to room temperature for 5 min.Cool to room temperature for 5 min.

Add 3µl of RNase solution and invert the Add 3µl of RNase solution and invert the tube 2-5 times.tube 2-5 times.

Incubate at 37°C for 15-60 min.Incubate at 37°C for 15-60 min.

Cool to room temp. for 5 min.Cool to room temp. for 5 min.

Add Add Protein Precipitation Protein Precipitation Solution(PPS)Solution(PPS)..

Vortex at high speed for 20 seconds.Vortex at high speed for 20 seconds.

Incubate in Ice for 5 min.Incubate in Ice for 5 min.

Centrifuge at 15,000 rpm for 3 min.Centrifuge at 15,000 rpm for 3 min.

Transfer the supernatant to a Transfer the supernatant to a microcentrifuge tube w/ 600 µl microcentrifuge tube w/ 600 µl

Isopropanol(IPA).Isopropanol(IPA).

Gently mix by inversion.Gently mix by inversion.

Centrifuge at 15,000 rpm for 3 min.Centrifuge at 15,000 rpm for 3 min.

Pour off the supernatant and drain the Pour off the supernatant and drain the tube on clean absorbent paper(Do not tube on clean absorbent paper(Do not

dry out the pellet)dry out the pellet)

Add 600µl 70% ethanol and invert the Add 600µl 70% ethanol and invert the tube several times to wash the DNA tube several times to wash the DNA

pellet.pellet.

Centrifuge at 15,000 rpm for 3 min.Centrifuge at 15,000 rpm for 3 min.

Pour off the Ethanol and drain the tube Pour off the Ethanol and drain the tube on clean absorbent paper.on clean absorbent paper.

Air dry the pellet for 10-15 min.Air dry the pellet for 10-15 min.

Add 100µl DNA Add 100µl DNA Rehydration Rehydration Solution(RH)Solution(RH)..

Incubate at 65°C for 1 hr. to rehydrate Incubate at 65°C for 1 hr. to rehydrate the DNA.the DNA.

Gently tap the tube to mix and store the Gently tap the tube to mix and store the DNA at 2-8°C.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

END!

Thank You for Listening!

References

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