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| |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> |
| |style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html> </html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}} | | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} |
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| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| ==Objectives== | | ==Objective== |
| # Make agar to plate petri dishes
| | Data analysis for reactions run on March 15 |
| # Make another set of serial dilutions
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| # Perform first disc diffusion assay
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| ==Materials== | | ==Data Workup protocol for all Main UV-VIS Machine analysis and Fluorescence Spectroscopy== |
| # Agar mix
| | UVVis |
| # Water | | #import data into excel |
| # 30 eppendorf tubes | | #Blank sample data by subtracting water blank absorbance values for before and after reaction |
| # Three 100mL falcon tubes | | #Absorbance against wavelength |
| # Protein-nanoparticle solutions | | # Monitor maximum absorbance wavelengths before and after |
| # Ampicilin
| | #monitor absorbance wavelengths |
| # 200µL pipette
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| # 2.8L flask
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| # Hot plate
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| # Two 1L screw-cap bottles
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| #Whatman Membrane Filter Paper (6mm discs punched out from larger discs) – autoclaved in dry cycle | |
| #Dilutions of Protein Nanoparticle Samples and Ampicillin Control
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| #Protein-Nanoparticle samples and Ampicillin Control Dilutions
| | Fluorescence |
| | | #import data into excel |
| AMP Table
| | #subtract water blank sample data from nanoparticle sample data (before and after) |
| {| {{table}}
| | #compute integral for each sample from between 305nm-470nm (peak size and cut off ends to avoid noise in integral calculation) |
| | align="center" style="background:#f0f0f0;"|'''Concentration (mg/L)'''
| | # calculate ratios of before and after of integrals for same same (just before and after) |
| | align="center" style="background:#f0f0f0;"|'''Concentration (ug/uL)'''
| | # monitor fluorescence peak emission wavelengths |
| | align="center" style="background:#f0f0f0;"|'''Volume (ul)'''
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| | align="center" style="background:#f0f0f0;"|'''Mass (ug)'''
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| | 4||0.004||25||0.1
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| | 8||0.008||25||0.2
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| | 16||0.016||25||0.4
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| |-
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| | 32||0.032||25||0.8
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| |-
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| | 64||0.064||25||1.6
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| |}
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| Example Nanoparticle Sample Dilutions
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|'''Concentration (x)'''
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| | 1
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| | 0.5
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| |-
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| | 0.25
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| |-
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| | 0.125
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| |-
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| | 0.0625
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| |}
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| # Agar disc diffusion specific dilution data, results and pictures can be found at the link [https://docs.google.com/spreadsheets/d/1XIncoYXZyIeGlpAFkbnr44U3PQ61jNbax-hkexNerUM/edit#gid=303734626 SRB Lab Notebook Data] for this entry's date
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| Disc Diffusion Procedure
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| # Thaw Ecoli frozen sample (from basement storeroom, retrieved with Dr. Hartings) for 30 min over ice
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| #Plate 100ul sample of thawed Ecoli solution onto mueller hinton agar plates by pipetting volume and then spreading with a triangular sterile spreader in a circle motion
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| #Load 20ul of sample dilutions or control dilution onto their own individual 6mm Whatman membrane filter paper discs with tweezers | |
| # place discs on plate, labeled so that dilution 1, is in a particular 5th of a plate (5 dilutions total) and immediately cover palte
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| # incubate at 37˚C overnight and check results after 24 hours incubation
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| Note that this technique was conducted on the benchtop. Between each loading of a disc on a plate, the tweezers used were washed with EtOH to minimize contamination. However, the plates were open to the air for a period of time during plating and loading of discs. Because we are testing for bacterial inhibition by the nanoparticles and control, if there is contamination, but inhibition we may never know that there was contamination in the first place. Discussed with Dr. Hartings and protocol was approved.
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| Checking the discs
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| # After 24hrs incubation at 37˚C remove discs from incubator and measure diameters of bacterial inhibition around the discs | |
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Project name
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