Template:SBB-Protocols Assay3: Difference between revisions
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====Treating with Silver==== | ====Treating with Silver==== | ||
#Make 10mM stock of AgNO3 | |||
#Add 200uL of 0.1 mM silver nitrate and resuspend the cells | #Add 200uL of 0.1 mM silver nitrate and resuspend the cells | ||
#Incubate overnight (24-48 hours) at room temperature | #Incubate overnight (24-48 hours) at room temperature | ||
#observe color change and precipitation of colored compound | #observe color change and precipitation of colored compound | ||
====other controls==== | ====other controls==== |
Revision as of 12:24, 15 April 2009
Silver Biomineralization Assay
Here we present an assay to determine whether the AG4 Silver binding peptide is present in the outer membrane of E.coli.
Procedure
Experimental Group
Group Samples
- 2x cell dilution. .1mM AgNO2. with arabinose.
- 2x cell dilution. .1mM AgNO2. without arabinose.
- 2x cell dilution. .05mM AgNO2. with arabinose.
- 2x cell dilution. .05mM AgNO2. without arabinose.
- 5x cell dilution. .1mM AgNO2. with arabinose.
- 5x cell dilution. .1mM AgNO2. without arabinose.
- 5x cell dilution. .05mM AgNO2. with arabinose.
- 5x cell dilution. .05mM AgNO2. without arabinose.
Picking and Incubating Colonies
- Add 4mL of LB media with the appropriate antibiotics to a clean test tube
- Pick a well-isolated, round, and "normal" looking colony of our proposed silver binding peptide with a pipet tip
- Drop it in the test tube
- Incubate at 37 overnight
Cleaning
- Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
- Dump supernatant, repeat to pellet another 1 mL (for a total of 2 mL)
- Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
- Add 200uL of TBS and resuspend
- Centrifuge the colony solution for 30 seconds
- Pour out the supernatant
- Add 200uL of TBS and resuspend
- Dilute the solution 10X. Measure OD of solution using spectrophotometer. (at 600nm)
Treating with Silver
- Make 10mM stock of AgNO3.
- Add 200uL of 0.1 mM silver nitrate and resuspend the cells
- Incubate overnight (24-48 hours) at room temperature
- observe color change and precipitation of colored compound
Control Group - non-AG4 cells
Picking and Incubating Colonies
- Add 4mL of LB media with the appropriate antibiotics to a clean test tube
- Pick a well-isolated, round, and "normal" looking colony of control E coli with no silver binding peptide with a toothpick
- Drop it in the test tube
- Incubate at 37 overnight
Cleaning
- Pellet 1 mL of saturated culture by spinning full speed, 30 seconds.
- Dump supernatant, repeat to pellet another 1 mL (for a total of 3 mL)
- Pour out the supernatant (contains extracellular proteins, unneeded nutrients)
- Add 200uL of TBS and resuspend
- Centrifuge the colony solution for 30 seconds
- Pour out the supernatant
- Add 200uL of TBS and resuspend
- Dilute the solution 10X. Measure OD of solution using spectrophotometer (at 600nm)
Treating with Silver
- Make 10mM stock of AgNO3
- Add 200uL of 0.1 mM silver nitrate and resuspend the cells
- Incubate overnight (24-48 hours) at room temperature
- observe color change and precipitation of colored compound
other controls
- Chemically reduce silver nitrate to precipitate silver (tells us silver nitrate is precipitating)
- Lyse cells over-expressing AG4 and run assay (tells us if AG4 is precipitating silver)
- Wash non-AG4 cells with silver nitrate and look for colour change (tells us if precipitation occurs without AG4)