User:James Chappell/ Notes

Project Protocols
Protocols being used by specific projects

Papers on steady state

 * The degradation rate of LuxR has been estimated by several experiments:
 * The LuxR was measured by western blot analyisis ~65minutes [1]
 * The LuxR rate of degradation is 0.7 hr-1 [2]

Optimizations
Mg2+ concentration is important. Optimum range is between 7-11mM.
 * Need to use a DNA extraction method that does not use ammonia. Recommend using sodium acetate rather than ammonia acetate to ethanol precipitate the plasmid.
 * Standard protocol uses temperatures of 37oC, however a shift to 24oC can prolong for 20 hours and a 2 fold increase in protein synthesis
 * NaCl of greater than 50mM NaCl can inhibit translation.
 * The level of DNA added should vary from 0.5ug to 4ug.

DNA Extraction generally increasing the volume increases the yield but decreases the concentration.
 * Cultures to be purified grown for no longer than 16hours.
 * Varying the elution buffer volume in the mini prep can vary the concentration and yield,
 * Host strains such as DH5alpha give good DNA extracts.

Protocol
Protocol: Day 1:

Preparation of Culture

 * 1) Placed 5 ml of LB in a 15 ml tube
 * 2) Added appropriate antibiotics into the tube
 * 3) Picked a colony from the fresh overnight plate
 * 4) Inoculated the colony in the LB media
 * 5) Grew upto 16hours at 37°C in shaking incubator
 * 6) *2x 15 Biobricks cloned