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| =August 2014=
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| ==06.08.2014 Revive Bacteria==
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| * revive bacteria strain [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#RP437_Strain RP437] from glycerol Calendar (see [[Christian Niederauer/Notebook/RacingBacteria/Calendar#Reviving_Bacteria_from_Glycerol_Calendar | Glycerol Protocol]])
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| * Master Plate is incubated ON, then stored in fridge
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| * Bacteria have to be checked for motility
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|
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| ==08.08.2014 PDMS Preparations==
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| [[Christian_Niederauer/Notebook/RacingBacteria/Calendar/Microfluidics/PDMS | PDMS Duplicate Protocol]]<br>
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| [[Christian_Niederauer/Notebook/RacingBacteria/Calendar/Microfluidics/Plasma_Cleaning | Plasma Cleaning Protocol]]<br>
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| Problems encountered with creating PDMS stamps:<br>
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| * oven is not entirely flat
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| * the "coring tool" is a cut glass pipette which continues to break and creates jagged holes
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| * device is prone to leakage if the holes are placed near the edge of the stamp
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| * devices are very thin and making it bigger by sticking additional pdms layer is tedious
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|
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| -> build [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Leveling_Petri_Dish leveling device], order coring tool, try cutting stamp so that the device is not near an edge, make deep araldite molds (thick stamp is hard to achieve with silicon wafer, as ideally the surface tension of the PDMS should keep the fluid on the wafer. If you add to much fluid, it will break surface tension and spread over the whole petri dish. Silicon wafer is too delicate to experiment with some ring-shaped devices to keep PDMS on the wafer.)
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|
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| ==11.08.2014 Checking for Motility==
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| * make suspension of one big and one small colony from the Master Plate created on [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#06.08.2014_Revive_Bacteria 06.08.] in eppi with 1mL PBS by picking it up with a micropipette tip and suspend tip into eppi
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| * cut window in double sided tape and put onto slide
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| * add a drop of bacteria suspension from the eppi(3µL) and put coverslide onto slide
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|
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| Result: bacteria from the big colony are motile, whereas bacteria from the small colony were not<br>
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| -> reinoculate bacteria from one or more big colonies of the master plate into liquid LB medium over night<br>
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|
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| As a comparison: 1µm latex beads on same coverslip-slide arrangement in 1:1000 dilution<br>
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| [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#13.08.2014_Tracking_Beads Analysis of Bead Movement]<br>
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|
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| Data: E:\12-08-rp437\ and E:\12-08-beads\
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|
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| ==12.08.2014 Reinoculate motile colony==
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| * put 5ml LB broth into falcon tube
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| * pick up one big colony with pipette tip and discard the tip in falcon tube
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| * incubate ON
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| * dip sterile inoculate ring into liquid colony and [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Streaking_Bacteria_onto_Agar_Plate streak] on agar plate the next day
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| * incubate plate over night => this is the new [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Master_Plate master plate]
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|
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| ==13.08.2014 Tracking Beads==
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| As a check, track beads based on [https://www.youtube.com/watch?v=dwnQW-FgbV4 microscopy images] acquired with Epitome, using BergLab tracking code. Brownian Motion is observed and after binning the frame-to-frame displacements, a rayleigh distribution is fit to the data with the curve fitting tool.
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| <gallery>Image:RaBa_tracks.png|tracked positions</gallery><br>
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| Matlab code for binning:<br>
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| ''botEdge=0; %first bin should start at 0 displacement''<br>
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| topEdge=25; %last bin should reach to 25''<br>
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| numBins=20; %number of bins''<br>
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| edges=linspace(botEdge,topEdge,numBins+1);'' <br>
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| binned=histc(disp,edges);''<br>
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| figure, plot(edges,binned,'.'); %plots the number of displacements which lie in each bin''
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| <gallery>Image:RaBa_rayleigh.png | distance in µm</gallery>
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|
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| ==25.08.2014 Araldite Replica from PDMS & COMSOL==
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| '''COMSOL Multiphysics'''<br>
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| Downloading COMSOL Multiphysics 4.3b 64-bit at the fedora linux machine in the dry lab.
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|
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| '''Araldite Replica'''<br>
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| According to the [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#How_to_create_a_Araldite_Mold_of_PDMS_Stamp_for_PDMS_Stamp Araldite Replica Protocol], 9g (5g of part A-resin and 4g of part B-hardener) are prepared, degassed and should have been poured after ~30 minutes. The break during the introduction course about the new microscope was after 1 hour. By then, the epoxy already was beginning to solidify. In addition to that, it seemed that the amount of epoxy mixed should be increased for easier pouring and handling (solidifies more slowly, more tolerance in terms of weight ratio).<br>
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| Manufacturing of replicas is postponed to 26.08.2014.
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|
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| '''Microscopy Course Notes'''<br>
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| * make new optical configuration: Configuration-> New Optical Configuration
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| * basic setting windows: View -> Acquisition Controls -> Clara Settings -> TI PAD, Sensor
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| * for DIC: ANALY & Condensor DIC N1 (20x,40x)
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| * always: [D] in, [NCB (neutral color balance)] in
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| * enable mouse focusing: Device -> Enable Mouse Joystick
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| * to save current window configurations: Save Current Layout as..
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| * Fluorescence: turn on NTNSL & filter 1 (blank with paper because shutter is not installed yet)
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| * high frame rate: no delay, exposure 1 frame, small ROI, Binning
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|
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| ==26.08.2014 ACTUAL Araldite Replica from PDMS & COMSOL==
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| '''Araldite Replica'''<br>
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| Using [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#How_to_create_a_Araldite_Mold_of_PDMS_Stamp_for_PDMS_Stamp Araldite Protocol]: Mixing 10g part A and 8g part B of Araldite and pouring onto the [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#H-Pattern_PDMS_stamps H-Pattern PDMS stamp] from 20.08.<br>
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| The replica will be called [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Araldite_Molds Araldite Replica I].<br>
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| Another Araldite replica is made with a pre-cut [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#H-Pattern_PDMS_stamps PDMS stamp made on 22.08.].<br>
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| An extra layer of PDMS is added below the stamp to achieve a deeper mold in the end. It is called [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Araldite_Molds Araldite Replica II].<br>
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| {| class='wikitable'
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| | <gallery>Image:RaBa_ara2.jpg | Araldite Replica II</gallery> || <gallery>Image:RaBa_ara2b.jpg | Araldite Replica II</gallery>
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| |}
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|
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| '''Comsol Multiphysics'''<br>
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| Always change into usr folder with ''cd ../''<br>
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|
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| Mount .iso with<br>
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| ''sudo mount -r -t iso9660 -o loop COMSOL43b_dvd.iso /media''<br>
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| ''cd media/''<br>
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| ''sudo sh setup''<br>
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|
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| Start License Server manually:<br>
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| ''cd local/comsol43b/license/glnxa64/''<br>
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| ''./lmgrd -c ../license.dat -l ../comsol44.log''
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|
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|
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| Run Comsol in ''local/comsol43b/bin/glnxa64/'' -> comsol<br>
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|
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| Gives error:<br>
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| <gallery>Image:RaBa_comsolerror.png</gallery>
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|
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| ==27.08.2014 PDMS from Araldite & Liquid Colony==
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| * with [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Preparation_of_PDMS_membrane PDMS-Protocol]: create PDMS from [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Araldite_Molds Araldite Replica II] and compare with [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#H-Pattern_PDMS_stamps original entire-pattern stamp from 20.08.].
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| * [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Inoculating_Bacteria_into_Liquid_Colony Liquid Colony is created] under hood with some left over sterile LB medium by discarding pipette tip into that tube after picking up a few colonys from the [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Master_Plate Master Plate].
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| * coring tools are too big (orange: 2.5mm, green: 3mm), have to wait for the right ones
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| Plan: punch holes into the [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#H-Pattern_PDMS_stamps PDMS from Araldite II], plasma clean with coverslip and attach tubings, then insert [http://en.wikipedia.org/wiki/Safranin safranin]. If it works properly, insert [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#Liquid_Colony bacteria suspension]<br>
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| '''Comparison of original PDMS and PDMS from Araldite''':<br>
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| "By eye" (i.e. with the dissection miscroscope), the two stamps look identical:
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| {| class='wikitable}
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| | <gallery>Image:RaBa_comparison1.JPG|Replica</gallery> || <gallery>Image:RaBa_comparison2.JPG| Original</gallery> || <gallery>Image:RaBa_comparison4.JPG|Replica </gallery> || <gallery>Image:RaBa_comparison3.JPG|Original </gallery>
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| |}
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| A proper comparison by measuring things will be done on the new microscope.
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|
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| ==28.08.2014==
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| ==29.08.2014==
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|
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| =September 2014=
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| ==01.09.2014==
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| ==02.09.2014==
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| ==03.09.2014==
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| ==04.09.2014==
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| ==05.09.2014==
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| ==08.09.2014==
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| ==09.09.2014==
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| ==10.09.2014==
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| ==11.09.2014==
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| ==12.09.2014==
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| ==15.09.2014==
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| ==16.09.2014==
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| ==17.09.2014==
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| ==18.09.2014==
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| ==19.09.2014==
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| ==22.09.2014==
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| ==23.09.2014==
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| ==24.09.2014==
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| ==25.09.2014==
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| ==26.09.2014==
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| ==29.09.2014==
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| ==30.09.2014==
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|
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| =October 2014=
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| ==01.10.2014==
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| ==02.10.2014==
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| ==03.10.2014==
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| ==06.10.2014==
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| ==07.10.2014==
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| ==08.10.2014==
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| ==09.10.2014==
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| ==10.10.2014==
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| =Stock=
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| ==Bacteria==
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| ====Master Plate====
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| Created on [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#12.08.2014_Reinoculate_motile_colony 12/08/2014] from liquid culture, which again was created from one motile colony of the original master plate.<br> The original master plate again was created from glycerol stock of [[Christian_Niederauer/Notebook/RacingBacterias/Calendar#RP437_Strain | RP437 E. Coli]] on [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#06.08.2014_Revive_Bacteria 06.08.].
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|
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| ====Liquid Colony====
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| *Created on 19/08/2014 ''[disappeared]''<br>
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| Suspended a few colonies from [[Christian_Niederauer/Notebook/RacingBacteria/Calendar#Master_Plate | Master Plate]] into 1ml of LB media.
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|
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| *Created on 27/08/2014<br>
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| Suspended a few colonies from [[Christian_Niederauer/Notebook/RacingBacteria/Calendar#Master_Plate | Master Plate]] into ~5ml of LB media.
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|
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| ====RP437 Strain====
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| [http://openwetware.org/images/0/01/RP437_bacteria_strain.pdf Datasheet of the RP437 Strain]
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|
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| After reviving this strain, test on soft agar plates and pick a colony that is motile and chemotactic because nonmotile variants arise in room temperature stab-cultures.
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|
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| ==Microfluidics Devices==
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| ====Leveling Petri Dish====
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| '''Problem:''' Oven is not even, the PDMS stamps therefore will be to thin at one side or the fluid even leaks over the waver and the stamp will be to thin everywhere.<br>
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| '''Solution:''' Filling up a Petri Dish with ~20ml PDMS and curing it in the oven (lower rack, placing dish at the very back on the right wall with the marker pointing perpendicular to the backwall).
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| The fluid's plane will be arranged perpendicular to the gravitational force eventually.
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| Future petri dishes with PDMS then can be placed on top of this device.
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|
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| ====H-Pattern PDMS stamps====
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| {|class="wikitable"
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| |-
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| | <gallery>Image:H_pattern.jpg | close up of 20.08. stamp</gallery> || <gallery>Image:H_pattern_wholes.jpg | problem: jagged holes, glass residues in stamp </gallery>
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| |}
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| *20.08. (entire pattern)
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| *22.08. (pre-cut)
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| *27.08. (Araldite Replica II)
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|
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| ====Various non- or bad-functioning racing tracks====
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|
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| ====Mother Machine Stamp (without holes / connections)====
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| ====Original Silicon Wafer with H-Pattern====
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| <gallery>Image:Silicon.jpg</gallery>
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| [http://openwetware.org/wiki/Image:RaBa_h_pattern.pdf Original Pattern etched on the Silicon Wafer]<br>
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| Track width: 2µm<br>
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| Track length: 362µm<br>
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| Number of tracks: 32
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|
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| ====Araldite Molds====
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| {| class='wikitable'
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| | <gallery>Image:RaBa_ara2.jpg | Araldite Replica II in the making </gallery> || <gallery>Image:RaBa_ara2b.jpg | Araldite Replica II</gallery>
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| |}
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| *Araldite Replica I, [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#26.08.2014_ACTUAL_Araldite_Replica_from_PDMS_.26_COMSOL created on 26.08.] from H-Pattern PDMS stamp (20.08.)
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| *Araldite Replica II, [http://openwetware.org/wiki/Christian_Niederauer/Notebook/RacingBacteria/Calendar#26.08.2014_ACTUAL_Araldite_Replica_from_PDMS_.26_COMSOL created on 26.08.] from H-Pattern PDMS stamp (20.08.) with increased thickness
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|
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| ==Material==
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|
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| *Coring Tool 2mm
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| *PDMS Chemicals (Sylgard, Silicone Elasteromer KIT 184)
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| * Araldite Chemicals (Resinova Bondtite)
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|
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| =Protocols=
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| ==Bacteria==
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| ====Important====
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| always work under hub (switch on Fan and Light), afterwards close hub and switch on UV<br>
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| Stuff contaminated with bacteria should be given to autoclaving room
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|
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|
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|
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| ===Streaking Bacteria onto Agar Plate===
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| * Petri dishes filled with 20ml of molten (80°C) 1.5% Agar, cool for 15 minutes
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| * with inoculator loop: dip into liquid culture of [[Christian Niederauer/Notebook/RacingBacteria/Calendar#Reviving_Bacteria_from_Glycerol_Calendar | revived bacteria]] and spread one line
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| * after initial line, burn inoculator ring & let it cool
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| * dip ring into initial line and spread bacteria diagonally
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| * burn ring again
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| * repeat ...
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|
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| <gallery>Image:Streak.gif | [http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/301labgraphics/streak.gif source]</gallery>
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|
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| ===Inoculating Bacteria into Liquid Colony===
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| * take micropipette with sterile tips and pick up a few colonies from your plate
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| * discard the tip into liquid LB medium
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| * incubate ON
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|
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| ===Reviving Bacteria from Glycerol Calendar===
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| * Scrape the frozen surface of the culture with a sterile inoculating needle, and then immediately [[Christian Niederauer/Notebook/RacingBacteria/Calendar#Streaking_Bacteria_onto_Agar_Plate | streak the bacteria]] that adhere to the needle onto the surface of an LB agar plate containing the appropriate antibiotics. Incubate the plate overnight at 37°C
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| * Scrape the frozen surface of the culture with a sterile inoculating needle or tip, and immediately immerse it in 2ml growth media within a 2059 snap-cap tube. Grow the bacteria overnight in a 37°C shaker.
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| * Return the frozen culture to storage at -70°C
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|
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| Source: [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4452 doi:10.1101/pdb.prot4452]
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|
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| ===Preparation of Glycerol Calendar for Long Term Storage of Bacteria===
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| * sterilize glycerol by autoclaving
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| * to 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol
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| * vortex the culture to ensure that the glycerol is evenly dispersed
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| * transfer the culture to a labeled storage tube
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| * freeze the culture directly to -70°C for long-term storage (up to 6 months)
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|
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| ==Microfluidics==
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| ===How to Create a PDMS-Stamp from Silicone/Araldite Mold===
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| Why PDMS? -> [http://en.wikipedia.org/wiki/Polydimethylsiloxane PDMS] is optically clear (good for microscopy!), and, in general, inert, non-toxic, and non-flammable.
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|
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| ====Preparation of PDMS mixture====
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| * with pipette boy and 5ml stripette pour ~4g of elastomer into a (clean) cup
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| * with micropipette (1000µl) add one tenth of elastomer mass (~0.4g) of linker fluid to the cup
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| * mix well with stripette
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| * degass for 30min-60min
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|
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| ====Preparation of PDMS membrane====
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| * pour degassed mixture onto wafer placed in petri dish with aluminium foil
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| * pour just enough to cover the waver
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| * use [[Christian Niederauer/Notebook/RacingBacteria/Calendar#Leveling_Petri_Dish | Leveling Petri Dish]] to ensure constant thickness
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| * araldite wafer: cure it for 3-4 hours in oven at 60°C
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| * silicon wafer: cure it for 2 hours in oven at 80°C for
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| * peel off membrane from wafer surface with forceps
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| * cut out the pattern but '''leave enough space around it'''
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|
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| ====Final Processing====
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| * punch holes with 2mm coring tool at the desired connection points under dissection microscope (tip: target the hole with coring tool under microscope, then put the stamp onto one finger with alufoil between stamp and finger and push/turn until you feel the coring tool on your skin)
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| * [[Christian Niederauer/Notebook/RacingBacteria/Calendar#Plasma_Cleaning | plasma clean]] the treated face of the stamp and a coverslip and press them together with alufoil in order to prevent contamination
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|
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|
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| ====Additional Procedures====
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|
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| '''If the stamp is too thin'''<br>
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| ('''DO BEFORE PUNCHING HOLES''')
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| * take a slice of PDMS with bigger or same area than the stamp
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| * [[Christian Niederauer/Notebook/RacingBacteria/Calendar#Plasma_Cleaning | plasma clean]] unshaped face of stamp and extra slice and stick them together
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| * now punch desired holes with 2mm coring tool
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|
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|
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| '''How to clean the PDMS cup'''
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| * scratch out all solid PDMS residues with forceps
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| * use tissue to clean out crudely
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| * pour pentane into the cup and gently sway the cup
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| * clean out with water and let dry
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|
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|
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| '''How to clean dirty stamp'''
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| * let the stamp soak in ethanol
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| * use forceps and (with caution) submerge the stamp into a jet of water
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| * let dry on clean alufoil
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|
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| ===Plasma Cleaning===
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| * turn on the dry air outside the building to 50 kg*m^-1*s^-2 (first open gas bottle valve by turning ccw, then turn black knob cw)
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| * turn on the dry air inside the building above the plasma cleaner to 30 (unit?) by turning cw
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| * turn on plasma cleaner, close all vents (vent ctrl, gas1 & gas2 by turning cw)
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| * settings: 70,30,10,1,restr,60,10,1,5,0
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| * add whatever you want to clean
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| * push start
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| * after "bleeding champer with gas" open gas 1 quickly to ~ 10^0 mbar by turning it ccw
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| * after "vent hold" open vent ctrl by turning it ccw
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| * after finishing process, close gas1 and vent ctrl again
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| * after end of lab work: close inside and outside gas valves
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|
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| '''Why Plasma Cleaning?'''<br>
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| PDMS has a hydrophobic surface because of a -CH_3 group present in its monomers. When oxidized with O_2 it reacts to Silanol (-SiOH) group which is hydrophilic. This group bonds with other hydrophilic groups (e.g. coverslip).
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|
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| '''Paramters in Detail for Plasma Cleaning:'''<br>
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| *Air inlet valve: 0.5kg/cm^2
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| *Power: 70W
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| *Ashing time: 20-30s
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| *Bleed delay time: 10s
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| *Gas 1 (left most valve): Set value to 1mbar
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| *Vent valve: restricted
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| *Restricted vent time: 120s
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| * Pump spin down time: 10s
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| *Vent hold time: 1s
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| *Gas shut time: 5s
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| *Turbo pumping No
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|
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| Reference: Chattopadhyay et al., 2005
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|
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|
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| ===How to create a Araldite Mold of PDMS Stamp for PDMS Stamp===
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| In order to minimize the use of the (expensive and delicate) silicon wafer, one can create a epoxy (Araldite) mold, which can be used in the same way as the silicon wafer, by the following steps:<br>
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| * in discardable container, mix Bondtite Araldite by taking weight ratio of 10:8 for resin(typ A) : hardener(typ B)
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| * mix thoroughly with (outside) pipette tip
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| * degass for at least 30mins
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| * pour onto PDMS mold inside petri dish wrapped in alufoil
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| * cure 2 days at RT or 3 hours at 50-60°C
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| * let cool afterwards and recollect the stamp (it is still usable)
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