Lab 5-Transformation

CaCl2

• Purpose- Makes E.coli cells able to take up the foreign DNA

• Ca2+ ions neutralize the negative charges on both the bacterial cell membrane and the plasmid DNA.

• This reduces electrostatic repulsion so DNA can get closer and bind the cell surface

CaCl₂ and ice first prepare competent cells by neutralizing negative charges on the bacterial membrane and stabilizing it. After DNA is added, CaCl₂ neutralizes both the DNA and membrane charges, reducing electrostatic repulsion and allowing the DNA to bind to the cell surface.

Ice incubation

• Stabilizes membrane and promotes DNA binding

• Cold temp makes the membrane more rigid which helps DNA stick to the surface of bac cells + preps cells for DNA uptake

Heat shock at 42 degrees

• used to drive DNA into the cell→ transformation

• Rapid temp change creates temporary pores in the membrane so DNA enters cytoplasm

TS broth

• Provides nutrients so bacterial cells can recover after heat shock (repair membrane damage) and express antibiotic resistance genes (ampicillin resistance)

Ampicillin (on plates)

• purpose: Selects for transformed cells→ tests whether bacteria carry amp resistance and can grow

• Ampicillin inhibits bacterial cell wall synthesis so only cells with the plasmid (carrying ampicillin resistance gene) survive

(TSA only plates control→ smear of colonies both with/without the plasmid)

What’s TE?

• colony forming units per ug of DNA used

• Transformation Efficiency =.Total number of transformed cells growing on the agar plate/ Amount of DNA spread on the agar plate (in µg)

  • measures how effectively the bacterial cells took up the foreign DNA

Add 10 uL of plasmid DNA to the re-suspended cells. Note: this is equivalent to 0.2 ug DNA (10 uL of a prepared stock at 1ug/50 uL).

5.3- Plasmid DNA Isolation: STET soln components

Sucrose

• Purpose- acts as an osmotic stabilizer, prevents cells from bursting prematurely

• creates a high osmotic environment outside the cell, which reduces water influx. This prevents cells from swelling and bursting before controlled lysis occurs, allowing the cell wall and membrane to be disrupted in a more controlled manner by other reagents (e.g., lysozyme and detergent).

Tris buffer

• Maintains stable pH, prevents DNA from chem degredation

EDTA

• Protects DNA from DNAses

• Binds Mg2+ ions which inhibits DNases that require these ions as cofactors to fx

Triton X-100

• Non-ionic detergent that disrupts lipid bilayers, solubilizes membranes, and causes controlled cell lysis, releasing cellular contents without harsh denaturation

• Non-ionic detergent that inserts its hydrophobic tail into the lipid bilayer, disrupting hydrophobic interactions and leading to membrane solubilization and gentle cell lysis without significant protein denaturation→ intact plasmid DNA, maintains integrity of DNA

• SDS harsher

Lysozyme

• Weakens bacterial cell wall by breaking B(1→4) glycosidic linkages in peptidoglycan which helps facilitate cell lysis

Phenol:Sevag

• Purpose- helps separate DNA from proteins and other debris

• Phenol denatures proteins making them insoluble in aq phase

• chloroform improves phase separation of the organic (phenol/chloroform) and aq layers, so proteins migrate to organic layer while DNA stays in aq layer

• Isoamyl alcohol helps reduce foaming and prevents emulsification→ cleaner seperation of layers

Sevag same thing removes residual phenol and proteins

KoAC

• precipitates contaminants by neutralizing charges causing these contaminants to aggregate

• helps remove proteins and detergents (in pellet) leaving plasmid DNA in aq supernatant

Isopropanol

• Precipiates DNA (after KoAC neutralized negative charges preventing repulsion)

• Lowers polarity of soln, weakens hydration shell, H20 less eff stabilizes DNA causes it to become more insoluble→ precip out to form pellet

70% ethanol

• Washes DNA

• removes salts (dissolve in water portion) while keeping DNA precipitated and insoluble bc ethanol still rel nonpolar

  
  

Pst1 (restriction enzyme)

• recognizes a specific palindromic DNA sequence and cleaves phosphodiester bonds within that sequence→ DNA fragments w/ sticky ends

PstI cuts like this:

5′–CTGCA↓G–3′
3′–G↑ACGTC–5

Restriction enzyme buffer

• Maintains the optimal chemical envt for restriction enzyme to fx in

• Provides optimal pH, ionic strength(conditions), and cofactors like Mg2+ for the enzyme to function

Loading dye

• Helps with gel loading and tracking

• adds density so sample sinks into wells, and contains dye to visualize migration

Agarose gel in TAE buffer

Agarose gel

• Seps DNA fragments

• DNA negatively charged moves toward + electrode

• smaller fragments move faster through gel matrix can navigate pores better, larger less far bc steric hindrance

TAE buffer

• Maintains stable pH and provides ions to conduct electric current during electrophoresis

SDS vs Triton X

Triton X-100 is preferred because it gently lyses cells without extensively denaturing proteins or releasing chromosomal DNA, allowing cleaner isolation of plasmid DNA compared to SDS.