Isolation of Genomic DNA from E. coli

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BACKGROUND

Isolation of genomic DNA is one of the most important and common experiment that is carried out in molecular biology and includes the transition from cell biology to molecular biology. The most common method of isolating genomic DNA without the use of commercial kit is by phenol/chloroform method. So, the basic objective of this test is to study the method of isolation genomic DNA from E.coli.

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REQUIREMENTS

Reagents and chemicals:   Tris base

Proteinase K

Phenol\chloroform (1: 1)

200 proof ethanol

RNAase

Ethanol

SDS

EDTA

Tryptone

Yeast extract

NaCl

LB medium (1% tryptone,0.5% yeast extract,200 mM NaCl)

TE buffer (10 mM Tris-Cl (pH 8.0),1 mM EDTA (pH 8.0))

Lysis buffer (10 ml)(9.34 ml TE buffer, 600 ul of 10% SDS ,60 μl of proteinase K (20 mg ml-1)).

Equipment:                        Tabletop centrifuge (Eppendorf)

1.5 ml Eppendorf tube

Incubator

Gloves

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PROCEDURE

At first take about 1.5 ml of E. coli overnight culture that was grown in LB medium and transfer it to a 1.5 ml of Eppendorf tube and then centrifuge it in maximum possible speed for about 1 min for extracting the cell pellet. Then discard the supernatant and resuspend the pellet in about 600 microliter of lysis buffer and completely vortex it for mixing it properly. Incubate it for about 1 hour at 37oC. To it add a equal volume of phenol/chloroform and properly mix it till they are mixed properly. Now at maximum speed spin for about 5 min which will lead to formation of a white layer in the aqueous phenol/chloroform interface.1 Now very carefully transfer the aqueous phase to a new tube through a 1ml pipette. You can repeat above two steps till the white layer disappears. For removing the phenol take equal volume chloroform and add to the aqueous layer. Mix it properly and spin at a maximum speed for about 5 min. Now transfer the aqueous layer to a new tube.2 For precipitation the DNA add about 2.5ml of cold ethanol and mix it properly. Precipitation may diffuse. For that you can keep the tube at -20oC for about 30 min and then spin it. You will be able to see DNA pellet.3 Now spin for about 15 min at 4oC. Discard the supernatant and rinse the DNA pellet with 1ml of 70% ethanol. Now again spin at maximum speed for about 2 min and discard the supernatant and wash the DNA pellet with 1ml of 70% ethanol. Now again spin at maximum speed. Now resuspend the DNA in TE buffer. Check genomic DNA on agarose gel.4

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CONCLUSION

It is a very simple method of isolating genomic DNA.

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REFERENCES

  1. Maniatis T., E.F. Fritsch, and J. Sambrook (1982). Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
  2. Blattner F. R., et al. (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474.
  3. Carnevale S., Velasquez J. N., Labbe J. H., Chertcoff A., Cabrera M. G., Rodriguez M. I.
  4. (2000) Diagnosis of Enter cytozoon bieneusi by PCR in stool samples eluted from filter paper disks. Clin. Diagn. Lab. Immunol.7:504–506.
  5. Fayer R., Lewis E. J., Trout J.M., Graczyk T.K., Jenkins MC., Higgins J., Xiao L., Lal A. A. (1999) Cryptosporidium parvum detection in oysters from commercial harvesting sites in the Chesapeake Bay. Emerg. Infect. Dis. 5:706–710.