Prokaryotic DNA isolation

on

 

Protocol: Phenol-chloroform extraction of prokaryotic DNA

  • Deoxyribonucleic acid ( DNA) extraction is a mechanism by which DNA is extracted from proteins, chromosomes, and other genetic material in the cell from which it is retrieved.
  • The smallest organisms, such as bacterial cells, are prokaryotes. Such prokaryotes consist of an outer lipid bilayer membrane and a cytoplasm containing a circular nucleus, inorganic salts, and metal ions proteins, sugar molecules, and other cell machine components.
  • There are a wide number of different methods for the efficient isolation of highly purified DNA from both eukaryotic and prokaryotic cells.
  • Most of the available DNA extraction methods have common elements. In fact, DNA extraction generally follows three basic steps:
  •  Lyse (break open) the cells. 
  • Separate the DNA from the other cell components. 
  • Isolate the DNA.

Phenol-chloroform extraction of DNA

The extraction of phenol-chloroform is a liquid-liquid extraction. Liquid-liquid extraction is a process that separates molecule mixtures based on the differential solubilities of the individual molecules in two different immiscible liquids. Liquid-liquid extraction is commonly used for the isolation of RNA, DNA, or proteins. Nucleic acid extraction requires applying an equal amount of phenol-chloroform to the aqueous solution of lysed cells or homogenized tissue, combining the two phases and allowing the phases to be separated by centrifugation. The centrifugation of the mixture results in two phases: the lower organic phase and the upper aqueous phase.

Principle

  1. The organism to be used should be grown in a favorable medium at an optimal temperature and should be harvested in the late log to early stationary phase for maximum yield.
  2. The genomic DNA isolation needs to separate total DNA from RNA, protein, lipid, etc.
  3. Initially, the cell membranes must be disrupted to release the DNA in the extraction buffer. SDS (sodium dodecyl sulfate) is used to disrupt the cell membrane.
  4. Once a cell is disrupted, the endogenous nucleases tend to cause extensive hydrolysis. DNA can be protected from endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a necessary cofactor for the action of most of the nucleases.
  5. Nucleoprotein interactions are disrupted with SDS, phenol, or proteinase K.
  6. Proteinase enzyme is used to degrade the proteins in the disrupted cell soup.
  7. Phenol and chloroform are used to denature and separate proteins from DNA. Chloroform is also a protein denaturant, which stabilizes the rather unstable boundary between an aqueous phase and a pure phenol layer.
  8. The denatured proteins form a layer at the interface between the aqueous and the organic phases which are removed by centrifugation.
  9. DNA released from disrupted cells is precipitated by cold absolute ethanol or isopropanol.

Materials and Reagents

  1. Tris base
  2. Proteinase K
  3. Phenol\chloroform (1:1)
  4. 200 proof ethanol
  5. RNAase
  6. Ethanol
  7. SDS
  8. EDTA
  9. Tryptone
  10. Yeast extract
  11. NaCl
  12. LB medium
  13. TE buffer
  14. Lysis buffer
  15. Tabletop centrifuge
  16. 1.5 ml Eppendorf tube
  17. Incubator

Procedure

  1. Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.
  2. Discard the supernatant without disturbing the cell pellet.
  3. Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend the cell pellet. 
  4. Incubate 1 h at 37 °C.
  5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
  6. Spin at max speed for 5 min at RT (all spins are performed at RT unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.
  7. Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipette (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).
  8. Steps 4-6 can be repeated until the white protein layer disappears.
  9. To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.
  10. Spin at max speed for 5 min.
  11. Remove aqueous layer to a new tube.
  12. To precipitate the DNA, add 2.5 or 3 volumes of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).
  13. Note: DNA precipitation may simply diffuse, which is normal. Keep the tube at -20 degrees for at least 30 min (the longer the better) and then spin it down (see Steps 15-16). You should see the DNA pellet. It looks transparent when it is wet and turns to white when it becomes dry.
  14. Incubate the tube at -20 °C for 30 min or more.
  15. Spin at max speed for 15 min at 4 °C.
  16. Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).
  17. Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.
  18. Resuspend DNA in TE buffer.
  19. Note: Large amounts of RNA will be present in the DNA sample. So, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to lysis buffer with a final concentration of 1 mg ml-1.
  20. Isolated Genomic DNA can be subjected to agarose gel electrophoresis.






Expected Results

  1. White strands of DNA precipitate.
  2. On gel, bands with smear patterns from high to low molecular weight range can be seen.
  3. Most of DNA fragments accumulated at high molecular weight: Not degraded.
  4. Most DNA fragments small: DNA might have got degraded.

References

  1. He, F. (2011). E. coli Genomic DNA Extraction. Bio-protocol Bio101: e97. DOI: 21769/BioProtoc.97.
  2. Maniatis T., E.F. Fritsch, and J. Sambrook (1982). Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Springs Harbor, NY.
  3. http://www.methodquarterly.com/2014/11/protocol-dna-extractions/
  4. https://genome.cshlp.org/content/4/6/368.full.pdf
  5. http://labcenter.dnalc.org/labs/dnaextraction/pdfs/teacher/Lesson%20Plan%20DNA%20Extraction%20from%20Bacteria.pdf
  6. https://www.sciencedirect.com/topics/neuroscience/dna-extraction

Comments

Post a Comment