Lesson 2 CRIPSR 241015

CRISPR Lesson 2

  1. Add DNA (plasmids) to ecoli
  2. Transformation -->

Shine light - OD600 - Opitcal Density at 600 nm - more ecoli, less light gets through

Solution - Lag phase: Starts clear, low OD600, not many ecoli cells - Log phase: Then exponential increases - Stationary Phase: Levels off, crowded, lack of nutrientients - Dead Phase: Lack of nutrients/crowded :: ecoli starts dying

Example dish: - ecoli - pink fluorcent - mfrp protein - picked a colony and put into a liquid culture - tied the colony to an antibiotic - cells are resistant to ampicillin - cells that don't uptake the dna die

Subculture - dilute and culture it again

p1000 - can pipette up to 100 -> 1000 microLiters

Lab: - LB - Luria Broth - Has nutrients that cells need to grow: amino acid, yeast extract for carbon source, salts, - Antibiotic - "carb" (carbonacillic) or "Ampacillian" - kills the microbe -

  • SBL001
    • strain of ecoli w/ added DNA (plasmid)
    • the plasmid is immune to the antibiotic
    • In a broth LB+carb and grown overnight
    • Will get rid of the plasmid