MCB 251 Midterm

Buffer P1

(1) Tris + EDTA + RNAase

Buffer P2

(2) Alkaline lysis

NaOH + SDS

Tris

Stabilizes pH in a physiological range. pH 7.5-8.5

Present in buffer P1, PE wash, EB buffer

EDTA

Sequesters divalent cations (Ca2+ and Mg2+) in order to slow DNA degradation by DNAases.

Destabilizes the outer membrane of cell, since the divalent cations serve as counterions for phosphate headgroups of phospholipid bilayer. The negative charges of the phosphate groups repel each other, destabilizing membrane.

Buffer P1

RNAase

Degrades RNA present in cell lysate so RNA does NOT co-purify with plasmid DNA.

Buffer P1

NaOH

Strong base that aids in membrane destabilization, breaking lipids into fatty acids.

Raises pH to a point where many proteins denature.

Denatures DNA, so large genomic DNA is no longer soluble in solution.

Buffer P2

Sodium Dodecyl Sulfate (SDS)

Detergent that intercalates into membrane, and in combination with high pH, ruptures cell.

Denatures proteins.

Buffer P2

Buffer N3

(3) GuHCl + potassium acetate

Denatures protein and prepares DNA for filter binding.

When N3 is added, a white soapy precipitate (detergent, denatured protein, chromosomal DNA) will form.

Plasmid DNA renatures and enters solution much faster than chromosomal DNA since it is smaller.

Potassium acetate

Buffer N3

Brings pH down to physiological range (pH 4.8)

Guanidine hydrochloride (GuHCl)

Chaotropic salt

Denatures proteins, specifically proteins that are capable of withstanding denaturation by detergent because they are bound to DNA (and not exposed to SDS).

Breaks solvation shell of DNA and forms salt bridges between DNA and silica column.

Helps immerse DNA and protein in solution.

Buffer PB

(4) Binding buffer that tightens silica membranes hold on DNA.

GuHCl + 30% isopropanol

GuHCl stabilizes DNA on silica column by keeping salt concentration high. Denatures proteins still bound to DNA, including NUCLEASES.

Isopropanol

Keeps DNA out of solution by creating a denaturing environment.

Buffer PB

PE wash

Tris + 80% ethanol

(5) Washes away GuHCl and potassium acetate to minimize amount of salts in eluted DNA.

Ethanol

PE wash

DNA is NOT soluble in high concentrations of ethanol. Without the ethanol, DNA would elute from column at this point.

Buffer EB

(6) Elution of plasmid

Tris - Buffers pH to alkaline pH where DNA is most stable

Lack of salt and ethanol will allow DNA to separate from column and renature —> elute with centrifugation.

3 kinds of plasmid isoforms

Open circle - Slowest, results from a nicked strand

Linearized - Middle of gel, indicative of mass, results from a double stranded break

Supercoiled - fastest

Plasmid

Extrachromosomal (doesn’t code for necessary cell functions like metabolism or replication) DNA molecule.

In bacteria: Usually covalently closed and circular, much smaller than host genomes.

Can confer survival advantage (antibiotic resistance)

High copy number plasmids

Unregulated or low stringency regulation mechanisms. Usually smaller and rely more on host replication machinery.

Gene product (e.g. protein) can lead to toxicity in host cell.

Low copy number plasmid

Strictly regulated replication mechanisms

Cloning vector

DNA from any organism into which a gene can be inserted.

Expression vector

Cloning vector.

Often have extra features leading to efficient transcription of mRNA of interest. Robust expression regulation to avoid toxicity.

Self-transmissible plasmids

Capable of transmitting themselves from one bacteria to another.

Plasmid encodes genes that enable self-transfer —> CONJUGATION

Forms mating pair with another recipient cell.

Restriction enzymes

Enzymes that cut double-stranded DNA at or near specific recognition sequences (restriction sites).

Bacteria and archaea.

Ligation

Stitching/tying together of DNA strands. Repairs phosphodiester backbone. DNA ligase.

Self-ligation

When the cloning vector becomes ligated to itself, making it so that the vector insert is NOT ligated.

Avoid this by increasing amount of insert DNA.

Concatenation

Multiple inserts are ligated together and inserted into vector.

To avoid - increase amount of vector DNA.