MCB 251 Buffers

What does P1 contain?

Tris, EDTA, and RNase A

What does P1 do?

Destabilizes outer membrane of bacterial cells

What does Tris do?

pH buffer that stabilizes the pH in a physiologic range

What does EDTA do?

it is a chelating agent and protects DNA from nucleases requiring a divalent cation, which will slow DNA degradation and stabilize the outer membrane of the cells

What does RNase A do?

it hydrolyzes RNA present in cell lysate, preventing RNA from co-purifying with the plasmid DNA

What does P2 contain?

NaOH and SDS

What does P2 do?

Lyses cellular membrane and denature proteins

What does NaOH do?

destabilize the membrane by chemically breaking some lipids into fatty acids and raise the pH to denature many proteins and DNA

What does SDS do?

it is a detergent that intercalates into the membrane, ruptures the cell, and denatures proteins

What does N3 contain?

potassium acetate and guanidine hydrochloride (GuHCl)

What does N3 do?

Precipitates bacterial cell contents (chromosomal DNA, proteins, membrane, etc..)

What does potassium acetate do?

it is a neutralizing buffer, bringing pH into physiologic range

What does GuHCl do?

it is a chaotropic salt that tends to denature proteins that are capable of withstanding denaturation by detergent, such as SDS from step 7, and helps to break down the solvation shell of the DNA and forms salt bridges between the DNA and the silica column

What does PB contain?

GuHCl and isopropanol

What does PB do?

Tightens DNA bound to silica in the column and wash membrane-bound DNA

What does isopropanol do?

it helps keep the DNA out of the solution by creating a denaturing environment.

What does PE contain?

ethanol (75-80%) and Tris

What does PE do?

Washes DNA bound to the silica membrane and washes away the GuHCl and potassium acetate, preventing too much salt in the eluted DNA

What would happen without ethanol in PE?

the DNA would elute from the column at this point

What does EB contain?

Tris

What does EB do?

Elutes DNA off of the silica membrane

What’s the order of adding these buffers?

P1, P2, N3, PB, PE, EB