Template:SBB-Protocols Assay3: Difference between revisions
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= Silver Biomineralization Assay = | = Silver Biomineralization Assay = | ||
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#Dump supernatant, repeat to pellet another 1 mL (for a total of 3 mL) | #Dump supernatant, repeat to pellet another 1 mL (for a total of 3 mL) | ||
#Pour out the supernatant (contains extracellular proteins, unneeded nutrients) | #Pour out the supernatant (contains extracellular proteins, unneeded nutrients) | ||
#Add | #Add <font color="darkturquoise">1 mL </font>of TBS (pH 7.4) and resuspend | ||
#Centrifuge the colony solution for 30 seconds | #Centrifuge the colony solution for 30 seconds | ||
#Pour out the supernatant | #Pour out the supernatant | ||
#Add 200uL of TBS and resuspend | #Add 200uL of TBS and resuspend | ||
#Dilute the solution 10X. Measure OD of solution using spectrophotometer (at 600nm) | #Dilute <font color="darkturquoise">an aliquot of </font>the solution 10X. Measure OD of solution using spectrophotometer (at 600nm) | ||
====Treating with Silver==== | ====Treating with Silver==== | ||
#Make 10mM stock of AgNO3 | #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 <font color="darkturquoise">JCA: Ammend this step. I think you want to say add 2uL of 10mM AgNO3 stock to 200uL of washed cells and vortext to mix (or something like that). </font> | ||
#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 | ||
Line 72: | Line 73: | ||
# Chemically reduce silver nitrate to precipitate silver (tells us silver nitrate is precipitating) | # 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) | # 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) | # Wash non-AG4 cells with silver nitrate and look for colour change (tells us if precipitation occurs without AG4)<font color="darkturquoise"> | ||
# Try growing any functional clones in cultures containing the AgNO3</font> |
Revision as of 09:31, 18 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
JCA: Is AgNO3 not AgNO2
Group Samples
- 2x cell dilution. 0.1mM AgNO3. with arabinose.
- 2x cell dilution. 0.1mM AgNO3. without arabinose.
- 2x cell dilution. 0.05mM AgNO3. with arabinose.
- 2x cell dilution. 0.05mM AgNO3. without arabinose.
- 5x cell dilution. 0.1mM AgNO3. with arabinose.
- 5x cell dilution. 0.1mM AgNO3. without arabinose.
- 5x cell dilution. 0.05mM AgNO3. with arabinose.
- 5x cell dilution. 0.05mM AgNO3. 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 37C 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 (pH 7.4) 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
- Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)
Control Group - non-AG4 cells
*note: do controls for each of the sample groups
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 1 mL of TBS (pH 7.4) and resuspend
- Centrifuge the colony solution for 30 seconds
- Pour out the supernatant
- Add 200uL of TBS and resuspend
- Dilute an aliquot of 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 JCA: Ammend this step. I think you want to say add 2uL of 10mM AgNO3 stock to 200uL of washed cells and vortext to mix (or something like that).
- Incubate overnight (24-48 hours) at room temperature
- observe color change and precipitation of colored compound
- Perform same procedure but vary concentration of AgNO3, cell density, and presence of arabinose (see group samples)
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)
- Try growing any functional clones in cultures containing the AgNO3