Team:Michigan/Alex's Notebook
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This does present another experiment idea, however: maybe we can try to culture MDAI2 alone, and then in co-culture with K12 to see if there it a difference in GFP. | This does present another experiment idea, however: maybe we can try to culture MDAI2 alone, and then in co-culture with K12 to see if there it a difference in GFP. | ||
At any rate, I'm not sure it's feasible to co-culture with algae, so quorum sensing project is probably done. | At any rate, I'm not sure it's feasible to co-culture with algae, so quorum sensing project is probably done. | ||
+ | |||
--[[User:Infekt|Infekt]] 17:25, 28 August 2010 (UTC) | --[[User:Infekt|Infekt]] 17:25, 28 August 2010 (UTC) | ||
+ | |||
+ | == 9/12/10 == | ||
+ | |||
+ | ''Alex'' | ||
+ | |||
+ | Made E. coli K12 broth culture from plate | ||
+ | *2 mL LB in 15mL tube | ||
+ | *37°C, 200 rpm shaking - ERB 1230 | ||
+ | Uploaded NanR cloning [[media:NanR_Cloning.pdf|protocol]] | ||
+ | |||
+ | --[[User:Infekt|Infekt]] 05:35, 13 September 2010 (UTC) | ||
+ | |||
+ | == 9/29/10 == | ||
+ | ''Alex and John'' | ||
+ | |||
+ | Dissolved M9 salts to 50X | ||
+ | *All of component A in 1000 mL DIwater | ||
+ | *All of component B in 1000 mL DIwater | ||
+ | Autoclaved both: each split into two 1000mL bottles filled w/ 500mL each (four bottles total) | ||
+ | *30 min sterilize time | ||
+ | Began biofilm assay following [[Media:Static+biofilm+quantification.pdf|Alex's biofilm assay protocol]] | ||
+ | |||
+ | Made broth cultures of ''P. putida'' LD1, ''P. fluorescens'' LD2, and LD1+LD2 coculture from plates, and ''P. putida'' KT2440 from Lin Lab frozen stock | ||
+ | *KT2440 inoculated into 2 mL LB in 15mL tube | ||
+ | *others inoculated into 2 mL LB + 100μg/mL amp in 15mL tube | ||
+ | *all incubated in 30°C, 200rpm shaking -- ERB 1230, 8:45pm | ||
+ | Streaked KT2440 on LB plate from frozen | ||
+ | *incubated 37°C -- ERB 1239 | ||
+ | Added KT2440 to strain database | ||
+ | Mixed M9 A and B solutions and diluted 1/50 in 200 mL sterile DIwater -- in 4 50mL tubes | ||
+ | *each tube 1 mL M9A, 1 mL M9B and 48 mL water | ||
+ | ---- | ||
+ | ''Alex'' | ||
+ | |||
+ | Uploaded [[Media:Static+biofilm+quantification.pdf|Alex's biofilm assay protocol]] | ||
+ | |||
+ | Added borax to two of the M9 tubes (100 mL) to buffer at ~pH 9.2 | ||
+ | - (.01 mol/L)*(.05 L)*(381.37 g/mol) = 190.7 mg borax/50 mL water | ||
+ | *Tested pH with pH indicator strips | ||
+ | **~9 | ||
+ | *Vacuum-filtered 100 mL w/ steriflips | ||
+ | |||
+ | == 9/30/10 == | ||
+ | ''Alex'' | ||
+ | |||
+ | Aliquot M9 and buffered M9 into five 15mL tubes each | ||
+ | Need 15 mL stocks of each of the following media -- each 15 mL neutral and 15 mL buffered at pH 9: | ||
+ | *M9 | ||
+ | *M9 + 0.4% glucose | ||
+ | *M9 + Acros NAs (250mg/L) | ||
+ | *M9 + Sigma Aldrich NAs (250mg/L) | ||
+ | *M9 + both Acros & Sigma NAs (each 250mg/L) | ||
+ | *LB | ||
+ | Added 150 μL 40% glucose to appropriate tubes (.4% final concentration) | ||
+ | Aliquot 15 mL LB into each of two 15mL tubes | ||
+ | Added NAs to appropriate tubes | ||
+ | - Acros: (.25 mg/mL)*(15mL)/(.91 mg/μL) = '''4.12 μL''' | ||
+ | - Sigma: (.25 mg/mL)*(15mL)/(.92 mg/μL) = '''4.08 μL''' | ||
+ | *added 4.1 μL of each NA to each respective tube | ||
+ | Added borax to one tube of LB to buffer at pH9 | ||
+ | - (190.7 mg)*(15μL/50μL) = '''57.2 mg''' per 15mL LB | ||
+ | *tested pH with indicator strip | ||
+ | **~9 | ||
+ | Tested pH of neutral M9 with indicator strip | ||
+ | *~7 | ||
+ | Moved KT2440 LB plate from 37°C to 4°C -- ERB 1239 | ||
+ | |||
+ | Continued [[Media:Static+biofilm+quantification.pdf|Alex's biofilm assay protocol]] | ||
+ | *made one 96-well plate for all strains and media at pH7 only | ||
+ | *plate template : | ||
+ | [[image:9-30 plate template.jpg]] | ||
+ | **100 μL/well: 99 μL medium + 1 μL culture | ||
+ | **covered plate with gas-permeable membrane | ||
+ | *Started 24-hr kinetic reading in Lin Lab microplate reader | ||
+ | **30°C | ||
+ | **OD600 absorbance | ||
+ | **read every 30 min | ||
+ | Made broth cultures for LD1, LD2 and KT2440 from yesterday's overnight broths | ||
+ | *2 mL each in a 15mL tube | ||
+ | *30°C, 200 rpm shaking - '''6:20pm''' | ||
+ | Discarded yesterday's overnight broths | ||
+ | |||
+ | --[[User:Infekt|Infekt]] 00:57, 1 October 2010 (UTC) | ||
+ | |||
+ | == 10/1/10 == | ||
+ | ''Alex'' | ||
+ | |||
+ | Moved broth cultures 37°C to 4°C -- 1:30pm (19-hr culture) | ||
+ | Prepared biofilm assay plate for all pH9 tests - following [[Media:Static+biofilm+quantification.pdf|Alex's biofilm assay protocol]] | ||
+ | *plate template: | ||
+ | [[image:9-30 plate template.jpg]] | ||
+ | **100 μL/well: 99 μL medium + 1 μL culture | ||
+ | *stored plate in Lin Lab 4°C -- 2:20pm | ||
+ | ---- | ||
+ | Saved data for curves from ph7 plate | ||
+ | Read pH7 plate ODs | ||
+ | Performed biofilm assay on ph7 plate | ||
+ | Started 24-hr kinetic reading for pH9 plate -- ~midnight | ||
+ | |||
+ | Discarded pH7 plate | ||
+ | |||
+ | Processed pH7 plate data | ||
+ | *uploaded results [[Media:Ph7_biofilm_assay_results.xls|here]] | ||
+ | |||
+ | --[[User:Infekt|Infekt]] 06:55, 2 October 2010 (UTC) | ||
+ | |||
+ | Uploaded pH7 kinetic data [[Media:Data_10-01-10-pH7_kinetic.txt|here]] | ||
+ | |||
+ | --[[User:Infekt|Infekt]] 04:06, 4 October 2010 (UTC) | ||
+ | |||
+ | == 10/3/10 == | ||
+ | ''Alex'' | ||
+ | |||
+ | Ran CV biofilm assay on pH9 plate, following [[Media:Static+biofilm+quantification.pdf|my protocol]] | ||
+ | *discarded plate | ||
+ | *uploaded [[Media:Ph9_biofilm_assay_results.xls|results]] | ||
+ | *uploaded [[Media:Data_10-01-10-pH9_kinetic.txt|pH9 kinetic raw data]] | ||
+ | |||
+ | --[[User:Infekt|Infekt]] 04:35, 4 October 2010 (UTC) |
Latest revision as of 04:36, 4 October 2010