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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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| ==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/Protocols#Reviving_Bacteria_from_Glycerol_Stock | 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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| <gallery>Image:Streak.gif | [http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/301labgraphics/streak.gif source]</gallery>
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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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| ==Reviving Bacteria from Glycerol Stock==
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| * Scrape the frozen surface of the culture with a sterile inoculating needle, and then immediately [[Christian Niederauer/Notebook/RacingBacteria/Protocols#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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| Source: [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4452 doi:10.1101/pdb.prot4452]
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| ==Preparation of Glycerol Stock 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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| =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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| ====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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| ====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/Stock#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/Protocols#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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| ====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/Protocols#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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| '''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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| '''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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| ==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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| Reference: Chattopadhyay et al., 2005
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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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