Week 6 Plasmid Miniprep Reagents

Reagents

What are the components of the lysis buffer?

Sugar, Tris buffer, Lysozyme, Detergent, and NaOH

What is the purpose of sugar in the lysis buffer?

After the bacterial membrane breaks, sugar enters and causes the cell to swell due to an osmotic pressure change

What is the purpose of Tris Buffer in the lysis buffer?

It maintains the pH and contains EDTA, which chelates divalent cations (Mg2+ and Ca2+) (taking them out of solution) to stop DNases from degrading the plasmid

What is the purpose of EDTA in Tris Buffer?

To chelate divalent cations (Mg2+ and Ca2+) (takes them out of the solution) to prevent DNases from degrading the plasmid

What is the purpose of Lysozyme in the lysis buffer?

To break down the cell walls of bacteria by destabilizing the peptidoglycan layer

Where is Lysozyme commonly found in humans?

In the saliva

What is the purpose of Detergent in the lysis buffer?

SDS and Triton X are the detergents

They lyse the cell membrane by destabilizing the lipopolysaccharide (LPS) layer

What are the detergents in lysis buffer?

SDS and Triton X

What is the purpose of NaOH in the lysis buffer?

It denatures the double stranded chDNA and plasmids by disrupting the H bonding between nitrogenous bases

What does the presence of NaOH do to the solution?

It makes the solution very basic/alkaline

What are the components of the neutralization buffer?

Glacial acetic acid, potassium acetate, RNase A, and Chaotropic Salt (Guanidinium Chloride)

What chaotropic salt is used in neutralization buffer?

Guanidinium chloride

What is the purpose of Glacial acetic acid in the neutralization buffer?

To neutralize the NaOH from the lysis buffer

As the pH nears neutrality, the plasmid renatures due to it's small circular shape

What happens to the plasmid and the chDNA in the presence of Glacial acetic acid?

The plasmid DNA renatures into two strands

The chDNA that is separated into ssDNA is too large to renature

What is the purpose of Potassium acetate in the neutralization buffer?

Potassium ions react with dodecyl sulfate (from SDS) to produce KDS (which is insoluble)

The KDS precipitates out of the solution with the lipids, proteins, and entangled chDNA (appearing as a white, goopy precipitate)

The Na+ ion from the SDS helps the plasmid bind to the silica membrane of the spin column

What is contained in the precipitate formed after the addition of neutralization buffer?

KDS, lipids, proteins, and entangled chDNA

What reacts with potassium ions to form KDS?

dodecyl sulfate from SDS

What is the purpose of RNase A in the neutralization buffer?

To remove any RNA remaining in the solution after lysis

What is the purpose of Guanidium Chloride in the neutralization buffer?

To maintain a hydrophobic environment by preventing H bonding between plasmids and water

To set up conditions to bind to the membrane

What are the components of the Wash buffer?

Wash buffer + 90% EtOH

What is the purpose of ethanol in the wash buffer?

Salts and small organic molecules go into solution with the ethanol (they are soluble in the ethanol), while the plasmid stays bound to the silica membrane

How are contaminants removed after adding wash buffer during the miniprep?

By centrifuging the spin column

What is the contents of the Elution Buffer?

DNA Grade water or TE Buffer

What is the purpose of Elution buffer?

To rehydrate the plasmid DNA and free it from the silica membrane

What are the steps of the plasmid miniprep?

Resuspension, lysis, neutralization, binding of DNA, Washing, then Elution