Genetics Lab 7 Quiz (Plasmid Miniprep Procedure)

1. Resuspension

2. Lysis

3. Neutralization

4. Washing

5. Elution

What are the 5 steps involved in the plasmid mini prep procedure?

Resuspend bacterial cells in a buffered solution

What is the purpose of resuspension?

RNase

What is the key ingredient of resuspension?

degrades RNA, ensuring the sample is free of RNA contamination

What is the purpose of RNase in resuspension?

cells are evenly resuspended and ready for lysis

What is the outcome of resuspension?

Break open bacterial cells to release cellular contents, including DNA

What is the purpose of lysis?

SDS and NaOH

What are the key ingredients of lysis?

A detergent that disrupts the cell membrane and denatures proteins

What is Sodium dodecyl sulfate (SDS) in lysis?

High pH denatures DNA and proteins

What is sodium hydroxide (NaOH) in lysis?

The bacterial cell lyse, and the mixture becomes viscous due to the release of macromolecules

What is the outcome of lysis?

neutralize the high pH and allow macromolecules to renature

What is the purpose of neutralization?

Potassium acetate

What is the key ingredient of neutralization?

Lowers pH, neutralizing NaOH

What is potassium acetate in neutralization?

Chromosomal DNA and proteins aggregate and precipitate, while plasmid DNA remains in solution, because plasmids are small, supercoiled, and renature correctly

What is the outcome of neutralization?

purify plasmid DNA by removing impurities

What is the purpose of washing?

Ethanol

What is the key ingredients of washing?

washing away salts, RNA, and other contaminants while plasmid DNA stays bound to the column

What does ethanol do in washing?

Purified plasmid DNA remains bound to the column; contaminants are washed away

What is the outcome of washing?

Release plasmid DNA from the spin column into solution

What is the purpose of Elution?

Elution buffer

What is the key ingredient of elution?

creates conditions where plasmid DNA no longer binds to the silica column

What does elution buffer do in elution?

Plasmid DNA is collected in the final elution solution

What is the outcome of elution?