Biotechnology: Principles & Processes — 5 Must-Know Facts — Summary

EcoRI cuts 5'-GAATTC-3' (palindrome) → produces sticky ends; from E. coli strain RY13; >900 restriction enzymes exist from 230+ bacterial strains
pBR322 markers = ampR + tetR; insertional inactivation into tetR identifies recombinants (grow on amp, not tet)
PCR temperatures: Denaturation 94–98 °C → Annealing 50–65 °C → Extension 72 °C; Taq polymerase from Thermus aquaticus (thermostable); 2ⁿ copies after n cycles
Gel electrophoresis: DNA is negatively charged → migrates to anode (+); smaller = farther; stained with ethidium bromide → orange under UV
Ti plasmid (Agrobacterium tumefaciens) must be disarmed for plant transformation; gene gun $\frac{gold}{tungsten particles}$ used for monocots; bacterial competency = $CaCl_{2}$ + 42 °C heat shock