DNA Structure and *E. coli* Overview

Study Notes

DNA is a complex organic molecule found in all prokaryotic and eukaryotic cells and many viruses.
It codes genetic information for inherited traits.
Each DNA strand is a long chain of monomer nucleotides.
Nucleotides consist of deoxyribose sugar, a phosphate group, and one of four nitrogenous bases: adenine (purine), guanine (purine), cytosine (pyrimidine), and thymine (pyrimidine).
Nucleotides are linked by covalent bonds between phosphate and sugar, forming a phosphate-sugar backbone with protruding bases.
Strands are held together by hydrogen bonds between bases: adenine with thymine, and cytosine with guanine.
The two strands are antiparallel.
The DNA double helix structure was discovered by James Watson and Francis Crick.

E. coli is a bacterium found in the lower intestines of warm-blooded organisms.
It's a facultative anaerobe (can survive with or without oxygen).
It's gram-negative: its cell wall has a thin peptidoglycan layer and an outer membrane.
The outer membrane acts as a barrier to some antibiotics.
E. coli stains pink with safranin (counterstain in Gram staining).
Most strains are harmless, but some cause food poisoning.

Step 1: Centrifuge E. coli culture to pellet cells.
Step 2: Discard supernatant carefully.
Step 3: Resuspend pellet in GTE buffer.
Step 4: Add lysis buffer (containing 1% SDS) to break open cells.
Step 5: Mix gently and incubate for 2 minutes.
Step 6: Add chilled isopropanol to precipitate DNA.
Step 7: Mix until DNA is visible as a white, stringy precipitate ("DNA spooling").
Step 8: Collect DNA using a glass rod or wooden stick.

Luria Broth (LB) medium: Tryptone (1.0g), yeast extract (0.5g), NaCl (0.5g), glucose (0.1g) in 100ml distilled water (pH 7.6).
GTE buffer: 50mM Glucose, 50mM Tris-HCl, 10mM EDTA.
10% SDS: 10g SDS in 100ml distilled water (pH 7.2). Do not autoclave.
70% Ethanol

Glucose (in GTE): Maintains osmolarity.
Tris-Cl (in GTE): Maintains pH, preventing DNA acid hydrolysis.
EDTA (in GTE): Chelates Mg²⁺ ions, inhibiting nucleases.
SDS (lysis buffer): Cell lysis and disruption of nucleoprotein interactions.
Isopropanol: DNA precipitation.

Extraction involves cell breakage and enzymatic inactivation of unwanted macromolecules.
Proteases and RNases inactivate proteins and RNA, respectively.
Genomic DNA appears as a white, gelatinous mass.
Extracted DNA can be stored frozen at -80°C for long-term preservation.