Christian Niederauer/Notebook/RacingBacteria/Protocols: Difference between revisions
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=Bacteria= | =Protocols= | ||
==Bacteria== | |||
====Important==== | ====Important==== | ||
always work under hub (switch on Fan and Light), afterwards close hub and switch on UV<br> | always work under hub (switch on Fan and Light), afterwards close hub and switch on UV<br> | ||
| Line 6: | Line 7: | ||
==Streaking Bacteria onto Agar Plate== | ===Streaking Bacteria onto Agar Plate=== | ||
* Petri dishes filled with 20ml of molten (80°C) 1.5% Agar, cool for 15 minutes | * Petri dishes filled with 20ml of molten (80°C) 1.5% Agar, cool for 15 minutes | ||
* with inoculator loop: dip into liquid culture of [[Christian Niederauer/Notebook/RacingBacteria/Protocols#Reviving_Bacteria_from_Glycerol_Stock | revived bacteria]] and spread one line | * with inoculator loop: dip into liquid culture of [[Christian Niederauer/Notebook/RacingBacteria/Protocols#Reviving_Bacteria_from_Glycerol_Stock | revived bacteria]] and spread one line | ||
| Line 16: | Line 17: | ||
<gallery>Image:Streak.gif | [http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/301labgraphics/streak.gif source]</gallery> | <gallery>Image:Streak.gif | [http://www.personal.psu.edu/faculty/k/h/khb4/enve301/301labs/301labgraphics/streak.gif source]</gallery> | ||
==Reviving Bacteria from Glycerol Stock== | ===Inoculating Bacteria into Liquid Colony=== | ||
* take micropipette with sterile tips and pick up a few colonies from your plate | |||
* discard the tip into liquid LB medium | |||
* incubate ON | |||
===Reviving Bacteria from Glycerol Stock=== | |||
* 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 | * 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 | ||
* 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. | * 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. | ||
| Line 23: | Line 29: | ||
Source: [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4452 doi:10.1101/pdb.prot4452] | Source: [http://cshprotocols.cshlp.org/content/2006/1/pdb.prot4452 doi:10.1101/pdb.prot4452] | ||
==Preparation of Glycerol Stock for Long Term Storage of Bacteria== | ===Preparation of Glycerol Stock for Long Term Storage of Bacteria=== | ||
* sterilize glycerol by autoclaving | * sterilize glycerol by autoclaving | ||
* to 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol | * to 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol | ||
| Line 30: | Line 36: | ||
* freeze the culture directly to -70°C for long-term storage (up to 6 months) | * freeze the culture directly to -70°C for long-term storage (up to 6 months) | ||
=Microfluidics= | ==Microfluidics== | ||
==How to Create a PDMS-Stamp from Silicone/Araldite Mold== | ===How to Create a PDMS-Stamp from Silicone/Araldite Mold=== | ||
Why PDMS? -> [http://en.wikipedia.org/wiki/Polydimethylsiloxane PDMS] is optically clear (good for microscopy!), and, in general, inert, non-toxic, and non-flammable. | Why PDMS? -> [http://en.wikipedia.org/wiki/Polydimethylsiloxane PDMS] is optically clear (good for microscopy!), and, in general, inert, non-toxic, and non-flammable. | ||
| Line 75: | Line 81: | ||
* let dry on clean alufoil | * let dry on clean alufoil | ||
==Plasma Cleaning== | ===Plasma Cleaning=== | ||
* 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) | * 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) | ||
* turn on the dry air inside the building above the plasma cleaner to 30 (unit?) by turning cw | * turn on the dry air inside the building above the plasma cleaner to 30 (unit?) by turning cw | ||
| Line 106: | Line 112: | ||
==How to create a Araldite Mold of PDMS Stamp for PDMS Stamp== | ===How to create a Araldite Mold of PDMS Stamp for PDMS Stamp=== | ||
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> | 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> | ||
* in discardable container, mix Bondtite Araldite by taking weight ratio of 10:8 for resin(typ A) : hardener(typ B) | * in discardable container, mix Bondtite Araldite by taking weight ratio of 10:8 for resin(typ A) : hardener(typ B) | ||
| Line 112: | Line 118: | ||
* degass for at least 30mins | * degass for at least 30mins | ||
* pour onto PDMS mold inside petri dish wrapped in alufoil | * pour onto PDMS mold inside petri dish wrapped in alufoil | ||
* cure 2 days at RT or | * cure 2 days at RT or 3 hours at 50-60°C | ||
* | * let cool afterwards and recollect the stamp (it is still usable) | ||
Revision as of 00:09, 27 August 2014
Protocols
Bacteria
Important
always work under hub (switch on Fan and Light), afterwards close hub and switch on UV
Stuff contaminated with bacteria should be given to autoclaving room
Streaking Bacteria onto Agar Plate
- Petri dishes filled with 20ml of molten (80°C) 1.5% Agar, cool for 15 minutes
- with inoculator loop: dip into liquid culture of revived bacteria and spread one line
- after initial line, burn inoculator ring & let it cool
- dip ring into initial line and spread bacteria diagonally
- burn ring again
- repeat ...
Inoculating Bacteria into Liquid Colony
- take micropipette with sterile tips and pick up a few colonies from your plate
- discard the tip into liquid LB medium
- incubate ON
Reviving Bacteria from Glycerol Stock
- Scrape the frozen surface of the culture with a sterile inoculating needle, and then immediately 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
- 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.
- Return the frozen culture to storage at -70°C
Source: doi:10.1101/pdb.prot4452
Preparation of Glycerol Stock for Long Term Storage of Bacteria
- sterilize glycerol by autoclaving
- to 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol
- vortex the culture to ensure that the glycerol is evenly dispersed
- transfer the culture to a labeled storage tube
- freeze the culture directly to -70°C for long-term storage (up to 6 months)
Microfluidics
How to Create a PDMS-Stamp from Silicone/Araldite Mold
Why PDMS? -> PDMS is optically clear (good for microscopy!), and, in general, inert, non-toxic, and non-flammable.
Preparation of PDMS mixture
- with pipette boy and 5ml stripette pour ~4g of elastomer into a (clean) cup
- with micropipette (1000µl) add one tenth of elastomer mass (~0.4g) of linker fluid to the cup
- mix well with stripette
- degass for 30min-60min
Preparation of PDMS membrane
- pour degassed mixture onto wafer placed in petri dish with aluminium foil
- pour just enough to cover the waver
- use Leveling Petri Dish to ensure constant thickness
- araldite wafer: cure it for 3-4 hours in oven at 60°C
- silicon wafer: cure it for 2 hours in oven at 80°C for
- peel off membrane from wafer surface with forceps
- cut out the pattern but leave enough space around it
Final Processing
- 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)
- plasma clean the treated face of the stamp and a coverslip and press them together with alufoil in order to prevent contamination
Additional Procedures
If the stamp is too thin
(DO BEFORE PUNCHING HOLES)
- take a slice of PDMS with bigger or same area than the stamp
- plasma clean unshaped face of stamp and extra slice and stick them together
- now punch desired holes with 2mm coring tool
How to clean the PDMS cup
- scratch out all solid PDMS residues with forceps
- use tissue to clean out crudely
- pour pentane into the cup and gently sway the cup
- clean out with water and let dry
How to clean dirty stamp
- let the stamp soak in ethanol
- use forceps and (with caution) submerge the stamp into a jet of water
- let dry on clean alufoil
Plasma Cleaning
- 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)
- turn on the dry air inside the building above the plasma cleaner to 30 (unit?) by turning cw
- turn on plasma cleaner, close all vents (vent ctrl, gas1 & gas2 by turning cw)
- settings: 70,30,10,1,restr,60,10,1,5,0
- add whatever you want to clean
- push start
- after "bleeding champer with gas" open gas 1 quickly to ~ 10^0 mbar by turning it ccw
- after "vent hold" open vent ctrl by turning it ccw
- after finishing process, close gas1 and vent ctrl again
- after end of lab work: close inside and outside gas valves
Why Plasma Cleaning?
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).
Paramters in Detail for Plasma Cleaning:
- Air inlet valve: 0.5kg/cm^2
- Power: 70W
- Ashing time: 20-30s
- Bleed delay time: 10s
- Gas 1 (left most valve): Set value to 1mbar
- Vent valve: restricted
- Restricted vent time: 120s
- Pump spin down time: 10s
- Vent hold time: 1s
- Gas shut time: 5s
- Turbo pumping No
Reference: Chattopadhyay et al., 2005
How to create a Araldite Mold of PDMS Stamp for PDMS Stamp
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:
- in discardable container, mix Bondtite Araldite by taking weight ratio of 10:8 for resin(typ A) : hardener(typ B)
- mix thoroughly with (outside) pipette tip
- degass for at least 30mins
- pour onto PDMS mold inside petri dish wrapped in alufoil
- cure 2 days at RT or 3 hours at 50-60°C
- let cool afterwards and recollect the stamp (it is still usable)
