Your question:

Big picture: Extract plasmid DNA from E. coli for transformation in lab 8 and eukaryotic high molecular weight from fruit

3. Repeat microfuge and vortex to disrupt pellet of cells and facilitate cell-resuspension in the next step

a. Why are you disrupting the formation of the pellet of cells?

7. Cells are resuspended from solution 1 in isotonic buffer

a Isotonic means same concentration of solute in the surrounding solution and solution inside the cell

b. NaOH raises pH of lysate and denatures DNA because of H bonding breaking L Plasmid DNA is supercoiled whereas genomic DNA is not causing genomic DNA to be broken down and plasmid DNA to remain

9. Solution 3 (acidic potassium acetate solution) is added and the solutions are mixed by inverting the tube

11. The pDNA and RNA are in the supernatant. The liquid is transferred to a clean tube and the pellet is discarded

12. The tube contains plasmids and RNA from millions of cells

Plasmid DNA Extraction Protocol Answers and Explanation:

Extracting Plasmid DNA from E. coli for Transformation: A Step-by-Step Guide

• Supernatant: Liquid remaining after centrifugation.

• pDNA: Plasmid DNA.

• Lysate: Cell contents released after lysis.

Step 1: Separating Cells from Medium (50 words)

• Resuspend the pellet in STE buffer to wash away remaining medium and stabilize DNA.

• Centrifuge again and discard the STE buffer.

Step 4: Neutralization and Precipitation (80 words)

• Add solution 3 (acidic potassium acetate) and mix. This neutralizes the pH and precipitates genomic DNA and proteins.

• Centrifuge and discard the supernatant.

• Wash the pellet with 70% ethanol to remove salts and impurities.

• The extracted pDNA can be used for various downstream applications in Lab 8, including restriction enzyme digestion, ligation, and transformation into eukaryotic cells.

• The high molecular weight DNA extracted from fruit will likely require additional purification steps depending on the intended use.