User:James Chappell/ Notes
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 
- The LuxR rate of degradation is 0.7 hr-1 
- Manefield M, Rasmussen TB, Henzter M, Andersen JB, Steinberg P, Kjelleberg S, and Givskov M. . pmid:11932456.
- Balagaddé FK, You L, Hansen CL, Arnold FH, and Quake SR. . pmid:15994559.
- Ronen M, Rosenberg R, Shraiman BI, and Alon U. . pmid:12145321.
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1482501 http://www.che.caltech.edu/groups/fha/balagadde_supplemental.pdf http://www.duke.edu/~you/publications/You_nature2004.pdf http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed&cmd=Retrieve&list_uids=15159530
- 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
Mg2+ concentration is important. Optimum range is between 7-11mM.
- NaCl of greater than 50mM NaCl can inhibit translation.
- The level of DNA added should vary from 0.5ug to 4ug.
- 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,
generally increasing the volume increases the yield but decreases the concentration.
- Host strains such as DH5alpha give good DNA extracts.
- Remove reagents from storage
- Prepare AHL dilutions
- Add cell extract in the appropriate wells following the plate schematic
- Add the appropriate volume of purified DNA to the sample and wait for x minutes
- Add AHL dilutions to appropriate well.
- Vortex the cells gently and centrifuge 5 seconds to bring the reaction down to the bottom of the tube.
Preparation of Culture
- Placed 5 ml of LB in a 15 ml tube
- Added appropriate antibiotics into the tube
- Picked a colony from the fresh overnight plate
- Inoculated the colony in the LB media
- Grew upto 16hours at 37°C in shaking incubator
- 2x 15 Biobricks cloned
- Transfer 1.5 ml of bacterial cells to an eppendorf
- Pellet cells by centrifuging for 30 seconds at maximum speed
- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to an eppendorf
- Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
- Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times
- Centrifuge for 10 min at maximum speed
- Pipette the supernatant into the QIAprep spin column on top of the eppendorf
- Centrifuge for 1 min and discard the flow-through.
- Wash with 0.5 ml Buffer PB, centrifuge for 1 min, and discard the flow-through
[Note: This step is unnecessary for cells without nucleases (DH5α, XL1-Blue]
- Wash with 0.75 ml Buffer PE
- Centrifuging for 1 min and discard the flow-through
- Centrifuge for an additional 1 min to remove residual wash buffer
- Place the QIAprep column in a clean 1.5 ml microcentrifuge tube
- To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min