Dandekar & Chandler:Protocols

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 34: Line 34:
==Specific Pseudomonas Projects==
==Specific Pseudomonas Projects==
*[[Dandekar & Chandler:Casein growth experiments|Casein growth experiments]]
*[[Dandekar & Chandler:Casein growth experiments|Casein growth experiments]]
-
 
-
 
-
==Casein growth FYIs==
 
-
*1% casein medium is somewhat opaque, but this is not why OD cannot be used as a measure of growth
 
-
*As pseudomonas elastase/proteases break down casein, the turbidity in the tube increases as the bits of degraded protein accumulate
 
-
:-- WT PAO1 will turn the tube milk white and completely opaque after ~4 hours in fresh medium
 
-
:-- A LasR mutant should NOT show the milky tube phenotype! If it does, you have contamination
 
-
::''if you want to keep the casein solution you have made, be sure to keep a negative control to rule out contamination there''
 
-
*To enumerate growth, colony counts should be performed
 
-
 
-
==Casein media recipes ==
 
-
===1% casein medium===
 
-
- 1% Casein sodium salt from bovine milk [[http://www.sigmaaldrich.com/catalog/product/sigma/c8654?lang=en&region=US| from Sigma]]
 
-
 
-
- 1x PM medium or 1x M9
 
-
 
-
Stir at medium high speed until casein is dissolved (can take anywhere from 1-3 hours depending on how awesome your stir plate is)
 
-
 
-
Filter sterilize
 
-
:-- ''Note that filter surface area is the same size in the 250 ml and 500 ml filter sterilization units; because the filters will clog, it's better to aliquot into 250 ml or smaller bottles''
 
-
 
-
===PM Medium===
 
-
''for 1000 mL''
 
-
"requires flushing with argon''
 
-
 
-
800 ml ddH2O
 
-
25 ml 0.5M Na2HPO4
 
-
25 ml 0.5M KH2PO4
 
-
10 ml 10% (NH4)2SO4
 
-
10 ml concentrated base (recipe follows)
 
-
 
-
:- Bring volume to 1000 ml and pH to 6.8
 
-
:- Autoclave for 45 min
 
-
 
-
1. Pour PM into round flask, flush with argon gas for 30 min, close with rubber stopper, and place medium and 16 ml hungate tubes (including lids and rubber stopper) into anaerobic glove box
 
-
2. Pour 10 ml into hungate tubes, and close with rubber stopper and lid
 
-
3. Remove tubes from glove box and autoclave
 
-
4. Add carbon sources from sterile stock solutions (e.g., acetate and succinate) 
 
-
 
-
 
-
==Casein growth protocols==
 
-
===Starting casein cultures===
 
-
'''Day 1'''
 
-
:From frozen stock, streak to LB plate and grow O/N at 37°C
 
-
'''Day 2'''
 
-
:Pick a single colony and inoculate into LB + 5mM MOPs buffer
 
-
:Grow O/N at 37°C with shaking
 
-
'''Day 3'''
 
-
:Into an 18mm tube with 3 mL 1% casein, transfer 30 μL O/N LB culture
 
-
:Grow O/N at 37°C with shaking
 
-
'''Day 4'''
 
-
:Into fresh 1% casein, transfer 100 μL from Day 1 casein culture
 
==Specific Burkholderia Projects==
==Specific Burkholderia Projects==

Revision as of 21:26, 5 March 2014

Home        Contact        Internal        Protocols        Lab Members        Publications        Research        Talks       


Contents

Media

General Protocols

Specific Pseudomonas Projects

Specific Burkholderia Projects

Co-Culture Projects

Personal tools