Alex Zorychta at 16:26, 4 June 2012

Alex Zorychta — Mon, 04 Jun 2012 16:26:49 GMT

←Older revision		Revision as of 16:26, 4 June 2012
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	==Overview==		==Overview==

-	Detailed below is the procedure to ~~make CaCl<sub>2</sub>-competent stock~~ cells~~. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm The Department of Biology at Indiana University]~~.	+	Detailed below is the procedure to passage ''B. pertussis'' cells.

	==Materials==		==Materials==

Alex Zorychta: New page: ==Overview== Detailed below is the procedure to make CaCl₂-competent stock cells. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm Th...

Alex Zorychta — Mon, 04 Jun 2012 16:26:19 GMT

New page: ==Overview== Detailed below is the procedure to make CaCl2-competent stock cells. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm Th...

New page

==Overview==

Detailed below is the procedure to make CaCl2-competent stock cells. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm The Department of Biology at Indiana University].

==Materials==

* Plate of ''B. Pertussis''
* SSM Buffer with Proline and SSM Supplements
* Sterile Polyester-Tipped Swabs
* Sterile Hood
* 25 mL sterile plastic pipette
* 37°C Water Bath
* 50 mL Erlenmeyer Flasks

'''Note: Media is stored in the refrigerator, while supplements are stored in the freezer.'''
'''Note: Supplements are stored in the freezer, but once they are used they can be stored in the refrigerater, but must be used within a week.'''

==Procedure==

* Warm up the SSM Buffer and Proline and SSM Supplements in a 37°C water bath for around 30 min.
'''Move all materials to a sterile hood.'''

* Using a sterile 25 mL plastic pipette, add 15 mL of warm SSM media to (each) flask
* Next, pipette 150 μL of '''each''' supplement (Proline & SSM) into each flask.
* Swab the ''Pertussis'' plate with a sterile polyester-tipped swab and mix it into the flask. Do this again to ensure you have transferred bacteria into the flask. ''Try to get a visible amount of bacteria into the flask, so it is a little cloudy.''
* Re-cover the flask with foil.
* Replace used swabs into their original covering and discard them into the biohazard waste.
* Place the flasks in the INFORS Minitron shaker/incubator (35.5°C and 150 RPM). Press Start.
* Incubate for 1 day. -- Later, measure at 650 nm wavelength.
* Wipe down the bench and hood with Cavicide and then Ethanol.

==Notes==
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!



==References==


==Contact==
*Who has experience with this protocol?

or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].


[[Category:Protocol]]

IGEM:Virginia 2012/Protocols/Passaging Pertussis - Revision history

Alex Zorychta at 16:26, 4 June 2012

Alex Zorychta: New page: ==Overview== Detailed below is the procedure to make CaCl2-competent stock cells. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm Th...

Alex Zorychta: New page: ==Overview== Detailed below is the procedure to make CaCl₂-competent stock cells. This is adapted from [http://sites.bio.indiana.edu/~chenlab/potocols/CompetentCellStock.htm Th...