Difference between revisions of "Template:BioE140LSpr09-Silver"
| Line 41: | Line 41: | ||
14. Pellet the cells <br> | 14. Pellet the cells <br> | ||
15. Measure absorbance of supernatant.The supernatant was tested for absorbance using spectroscopy on a TECAN set to measure the wavelength range 350nm-750nm at 10nm intervals. | 15. Measure absorbance of supernatant.The supernatant was tested for absorbance using spectroscopy on a TECAN set to measure the wavelength range 350nm-750nm at 10nm intervals. | ||
| − | [[Image: | + | [[Image:silverbinding.jpg]] |
Revision as of 19:25, 11 May 2009
Silver Binding Assay
Silver Binding Assay – Characterization of AG4 composite parts
Information
Control: pBca9145-Bca1363
11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}<br>
12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}<br>
13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}<br>
14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}<br>
15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}<br>
16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}<br>
17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}<br>
LB: ampicillin and kanamycin
Conditions: pH 7.4 TBS. Ambient light. Constant agitation. Silver Nitrate (AgNO3) does not react with LB or TBS at dilute concentrations.
Protocol
Pick and Incubate Colonies
1. Add 4mL of LB media with the appropriate antibiotics and arabinose
2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a pipet tip
3. Drop it in the test tube
4. Incubate at 37C overnight
Wash cells and incubate in AgNO3
5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)
7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
8. Add 200uL of TBS (pH 7.4) and resuspend
9. Centrifuge the colony solution for 30 seconds
10. Pour out the supernatant
11. Add TBS and 0.1mM AgNO3, and resuspend
12. Incubate at room temperature for 24hours
13. Observe color
Measure Absorbance using Spectrophotomer
14. Pellet the cells
15. Measure absorbance of supernatant.The supernatant was tested for absorbance using spectroscopy on a TECAN set to measure the wavelength range 350nm-750nm at 10nm intervals.
