Lab Practical Flashcards
Plasmid DNA Isolation - Key Steps
Resuspension: Cells suspended in buffer.
Lysis: SDS & alkaline buffer disrupt cells.
Neutralization: Precipitates chromosomal DNA.
Purification: Plasmid DNA remains in supernatant.
Silica-based Extraction: DNA binds, is washed, and eluted.
Role of SDS in Plasmid Isolation
Sodium Dodecyl Sulfate (SDS): Anionic detergent.
Disrupts cell membranes by breaking lipid bilayers.
Denatures proteins & chromosomal DNA.
Allows plasmid DNA to remain intact in solution
Agarose Gel Electrophoresis - Process
Prepare gel: Mix agarose & buffer, heat, pour.
Load samples: Add DNA with loading buffer.
Apply current: DNA moves toward +ve anode.
Visualize: Use UV & ethidium bromide.
Plasmid DNA Forms & Migration
Supercoiled: Tight, migrates fastest.
Open Circular: One nick, migrates slower.
Linear: Both strands cut, medium migration.
Key Electrophoresis Reagents
Agarose: Forms gel matrix for separation.
TAE/TBE Buffer: Maintains pH & conductivity.
Ethidium Bromide: Binds DNA, fluoresces under UV.
Loading Buffer: Adds density & tracking dye.
1 kb Ladder: DNA size reference.
Hazards & Safety in Gel Electrophoresis
SDS & Ethidium Bromide: Toxic, wear gloves.
Boiling Agarose: Can cause burns.
Electric Shock: Avoid touching live apparatus.
UV Radiation: Protect eyes & skin.
Plasmids - Key Facts
Small, circular DNA molecules in bacteria.
Replicate independently of chromosomal DNA.
Can carry antibiotic resistance or other beneficial genes.
Transferred between bacteria via conjugation or transformation.
Plasmid Cloning Vectors - Features
Origin of replication – Ensures high copy number.
Antibiotic resistance gene – Selects for plasmid-containing cells.
Multiple cloning site (MCS) – Contains restriction enzyme sites.
Bacterial Culture for Plasmid Isolation
LB medium: Nutrient-rich broth for bacterial growth.
Selective antibiotic: Ensures only plasmid-containing cells survive.
Overnight incubation: Bacteria multiply before DNA extraction.
Alkaline Lysis - How It Works
SDS: Breaks cell membrane, denatures proteins & DNA.
NaOH: Denatures chromosomal & plasmid DNA.
Neutralization buffer:
Precipitates chromosomal DNA.
Plasmid DNA stays in solution.
Spin Column DNA Purification - Steps
Plasmid binds to silica in high salt buffer.
Wash steps remove proteins & contaminants.
Elution with water or buffer releases pure DNA.
Agarose Gel - Key Points
0.7% gel → Large DNA fragments.
2% gel → Small DNA fragments.
0.8% gel used for plasmid separation
DNA Migration in Electrophoresis
DNA is negatively charged → Moves toward +ve anode.
Smaller fragments move faster through gel pores.
Supercoiled DNA moves faster than linear DNA.
Ethidium Bromide - Properties & Risks
Intercalates into DNA, fluoresces under UV light.
Mutagenic & toxic – Always wear gloves.
Disposal: Use dedicated hazardous waste bins.
