Difference between revisions of "BioE140LSpr09-Silver"
(New page: Silver Binding Assay – Characterization of AG4 composite parts Information Control: pBca9145-Bca1363 Composite Parts: 11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm} 12: {Pba...) |
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− | Silver Binding Assay – Characterization of AG4 composite parts | + | Silver Binding Assay – Characterization of AG4 composite parts <br> |
− | Information | + | Information<br> |
− | Control: pBca9145-Bca1363 | + | Control: pBca9145-Bca1363<br> |
− | Composite Parts: | + | Composite Parts: <br> |
− | 11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm} | + | 11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}<br> |
− | 12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm} | + | 12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}<br> |
− | 13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm} | + | 13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}<br> |
− | 14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm} | + | 14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}<br> |
− | 15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm} | + | 15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}<br> |
− | 16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm} | + | 16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}<br> |
− | 17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm} | + | 17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}<br> |
+ | <br> | ||
+ | LB: ampicillin and kanamycin<br> | ||
− | + | Conditions: pH 7.4 TBS. Ambient light. Constant agitation. Silver Nitrate (AgNO3) does not react with LB or TBS at dilute concentrations.<br> | |
− | + | Protocol<br> | |
− | + | ||
− | Protocol | + | |
==Pick and Incubate Colonies== | ==Pick and Incubate Colonies== | ||
− | 1. Add 4mL of LB media with the appropriate antibiotics and arabinose | + | 1. Add 4mL of LB media with the appropriate antibiotics and arabinose <br> |
− | 2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a | + | 2. Pick a well-isolated, round, and "normal" looking colony containing the silver binding peptide with a pipette tip<br> |
− | 3. Drop it in the test tube | + | 3. Drop it in the test tube<br> |
− | 4. Incubate at 37C overnight | + | 4. Incubate at 37C overnight<br> |
==Wash cells and incubate in AgNO3== | ==Wash cells and incubate in AgNO3== | ||
− | 5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds. | + | 5. Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.<br> |
− | 6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL) | + | 6. Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)<br> |
− | 7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients) | + | 7. Pour out the supernatant (contains extracellular proteins, unneeded nutrients)<br> |
− | 8. Add 200uL of TBS (pH 7.4) and resuspend | + | 8. Add 200uL of TBS (pH 7.4) and resuspend<br> |
− | 9. Centrifuge the colony solution for 30 seconds | + | 9. Centrifuge the colony solution for 30 seconds<br> |
− | 10. Pour out the supernatant | + | 10. Pour out the supernatant <br> |
− | 11. Add TBS and 0.1mM AgNO3, and resuspend | + | 11. Add TBS and 0.1mM AgNO3, and resuspend<br> |
− | 12. Incubate at room temperature for 24hours | + | 12. Incubate at room temperature for 24hours<br> |
− | 13. Observe color | + | 13. Observe color<br> |
==Measure Absorbance using Spectrophotomer== | ==Measure Absorbance using Spectrophotomer== | ||
− | 14. Pellet the cells | + | 14. Pellet the cells <br> |
15. Measure absorbance of supernatant | 15. Measure absorbance of supernatant |
Revision as of 00:56, 11 May 2009
Silver Binding Assay – Characterization of AG4 composite parts
Information
Control: pBca9145-Bca1363
Composite Parts:
11: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPG_L6!}{dblTerm}
12: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<eCPX!}{dblTerm}
13: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<upaG_short!}{dblTerm}
14: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<Ag43_short!}{dblTerm}
15: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<espP(beta)!}{dblTerm}
16: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<ehaB!]{dblTerm}
17: {Pbad.rbs.prepro.StrepTag}{<AG4>}{<CPompX!}{dblTerm}
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 pipette 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