Plasmid Preparation and DNA Extraction

Vocabulary terms and definitions covering bacterial genomic vs. plasmid DNA, alkaline lysis plasmid purification, and crude banana DNA extraction.

Bacterial genomic DNA

The circular DNA anchored to the plasma membrane that encodes essential functions of a bacterial cell.

Bacterial plasmid DNA

Small, circular DNA molecules that float freely in the cytoplasm, usually existing in a supercoiled conformation.

Plasmid genes

Genes not required for cell survival that confer non-essential attributes, such as allowing catabolism of unusual compounds or survival in harsh conditions.

p107

A specific plasmid that confers resistance allowing E. coli to grow in the presence of the antibiotic kanamycin.

Kanamycin

An antibiotic that disrupts bacterial protein synthesis.

High molecular weight (HMW) DNA

A designation used for eukaryote DNA due to its exceptionally large molecular size.

Plasmid prep

A laboratory method that separates bacterial genomic DNA from plasmid DNA on the basis of size and conformation.

Microcentrifuge (Microfuge)

A laboratory machine that generates centrifugal force to cause suspended bacterial cells or precipitates to pack into a pellet at the bottom of a tube.

Supernatant

The liquid phase found above the pellet.

STE

A wash solution used to rinse residual liquid growth medium off bacterial cells.

Solution I

A buffered isotonic solution used to resuspend bacterial cell pellets.

Solution II

Contains SDS and NaOH. SDS is a detergent which causes cell lysis, NaOH raises the pH and breaks hydrogen bonds in the DNA.

Solution III

An acidic potassium acetate solution that neutralizes the lysate, causing potassium to form an insoluble salt with SDS that co-precipitates with proteins, cell debris, and tangled genomic DNA.

95% ethanol (in plasmid prep)

An alcohol that removes water from DNA, allowing $Na^+$ ions to interact with the negative backbone and precipitate plasmid DNA.

70% ethanol (in plasmid prep)

A wash solution that dissolves and removes residual salts from the DNA pellet while slightly hydrating it to make the pellet easier to dissolve.

Tris Buffer (Solution T)

A buffer used to resuspend the final purified plasmid pellet; keeping it on ice prevents residual DNases from degrading the plasmid.

Dish soap (in banana DNA extraction)

A non-polar substance used to dissolve the lipid bilayer of cell membranes so cellular DNA becomes accessible.

Salt (NaCl, in banana DNA extraction)

A neutral salt whose ions neutralize the negative phosphate charges on DNA strands, enabling them to clump together.