Step I: Homogenization 1. Take g onion and make slices of 1-2 mm pieces 2. Obtai

Question

Step I: Homogenization 1. Take g onion and make slices of 1-2 mm pieces 2. Obtain a mass of 20 3. Add 100 ml homogenizing solution. gresh sliced onion in a 250 ml conical flask. 4. Mix thoroughly and incubate the flask at 65 C for 15 minutes What happens at this stage by: Heat (63 C): soften onion tissues, loosen cells, denature enzymes. SDS: dissolves membranes, and denatures protein. 5. Incubate the flask in ice bath (pieces of ice in water) to lower temperature to 20°C. 6. Pour cooled onion slice preparation in a blender and fasten the lid. Homogenize for 30 seconds at low speed, followed by 30 seconds at high speed. What happens by: Homogenization: Na-citrate: 7. Pour homogenate from the blender and keep on ice for 10 minutes. 25

Explanation / Answer

The onion tissue is mixed in a blender with the homogenization medium, which breaks down the cell wall, cell membrane, and nuclear membrane. The homogenization medium contains three important chemicals that allow the release of DNA. The first is sodium dodecyl sulfate or SDS. It is a detergent that helps to break down the cell membrane. It emulsifies the lipids and proteins of the cell by disrupting the non-covalent interactions that hold the cell membrane together. SDS also forms complexes with these lipids and proteins causing them to precipitate out of solution. Ethylenediamine tetracetic acid or EDTA weakens the cell by complexing with the divalent cations, Mg2+ and Ca2+, which are needed for membrane stability. Therefore, EDTA also aids in the lysing of the cells. Lastly, sodium chloride enables the DNA to precipitate out of an alcohol solution because it shields the negative charges on the phosphate groups of DNA. Thus, the strands will not electrostatically repel and will coalesce.

The NaCl/sodium citrate buffer stabilizes the DNA by forming a Na+ shell around the negatively charged phosphates of the DNA. The citrate inactivates the DNAse that would otherwise break down the DNA.

Related Questions

Navigate

• Browse (All)
• Browse S
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.