IGEM:Virginia 2012/Protocols/CaCl2 Competent Stock Cells - Revision history

Joseph J. Muldoon at 16:35, 4 June 2012

2012-06-04T16:35:03Z

←Older revision		Revision as of 16:35, 4 June 2012
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	* Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).		* Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).

-	* Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of .~~6A600~~ is achieved; put on ice and keep chilled at 0ºC for 15 min.	+	* Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of A600=0.6 is achieved; put on ice and keep chilled at 0ºC for 15 min.

	* Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.		* Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.

-	* Resuspend cells with 15 mL sterile, ice-cold .1M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave on ice 15 min. Spin again at 4000 rpm for 10 min.	+	* Resuspend cells with 15 mL sterile, ice-cold 0.1M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave on ice 15 min. Spin again at 4000 rpm for 10 min.

	* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).		* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).

Alex Zorychta: /* Overview */

2012-06-04T14:30:36Z

Overview

←Older revision		Revision as of 14:30, 4 June 2012
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	==Overview==		==Overview==

-	Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells.	+	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].

	==Materials==		==Materials==

Alex Zorychta: /* Procedure */

2012-06-04T14:29:42Z

Procedure

←Older revision		Revision as of 14:29, 4 June 2012
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	* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).		* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).

-	**Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).	+	*Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).

	* Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.		* Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.

Alex Zorychta at 14:29, 4 June 2012

2012-06-04T14:29:23Z

←Older revision		Revision as of 14:29, 4 June 2012
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	==Overview==		==Overview==

-	~~This~~ is ~~how~~ to ~~isolate the phage genome from a preparation of bacteriophages, specifically the ''Bordetella'' phage~~.	+	Detailed below is the procedure to make CaCl<sub>2</sub>-competent stock cells.

	==Materials==		==Materials==

-	* ~~10 mM EDTA~~	+	* 5 mL LB Media
-	* ~~CsCl-banded phage~~	+	* 200 mL LB Media
-	* ~~sterile tube~~	+	* Ice
-	* ~~100% ethanol~~	+	* Four 50 mL sterile centrifuge (conical) tubes
-	* -20°C freezer	+
	* Centrifuge		* Centrifuge
-	* 0.~~5 mL of 70% ethanol~~	+	* 0.1 M CaCl<sub>2</sub>
-	* ~~TE Buffer~~	+	* 0.1 M CaCl<sub>2</sub> / 15% glycerol

	==Procedure==		==Procedure==

-	* Add 10 volumes of 10mM EDTA pH 8.~~0 to 1 volume of CsCl-banded phage in a sterile tube~~	+	Day 1: Plate cells and grow overnight.
-	* Heat for 5 minutes at 65°C; let cool to room temperature	+
-	* Add 2.~~5 volumes 100% ethanol; place in -20°C freezer for 20 minutes~~	+	Day 2: Entire procedure should take approximately one hour.
-	* ~~Centrifuge: 10,000g for~~ 5 ~~minutes~~	+
-	* ~~Discard supernatant; rinse pellet~~ with 0.~~5ml 70% ethanol~~; ~~centrifuge again; discard supernatant; rinse pellet with 0~~.~~5ml 70% ethanol; centrifuge again; discard supernatant~~	+	* Pick single colony and grown in 5 mL LB media for 2-3 hrs with vigorous shaking (~300rpm).
-	* ~~Let pellet air dry~~ at ~~room temperature~~	+
-	* Resuspend ~~DNA in TE buffer; let DNA sit for several hours to go into solution; or~~, ~~restriction digests can be performed immediately; store solution of DNA at~~ -~~20°C for long-term storage~~ (i.e. ~~months) or~~ at ~~4°C~~ for ~~short~~-~~term storage (i.e~~. 1-~~2 days~~)	+	* Transfer into 200 mL LB media and grow cells with vigorous shaking until an absorbance of .6A600 is achieved; put on ice and keep chilled at 0ºC for 15 min.
		+
		+	* Apply cells into four 50 mL sterile centrifuge (conical) tubes and spin cells at 4000 rpm for 10 min.
		+
		+	* Resuspend cells with 15 mL sterile, ice-cold .1M CaCl2 by gentle pipetting (DO NOT VORTEX). Leave on ice 15 min. Spin again at 4000 rpm for 10 min.
		+
		+	* Resuspend in 4 mL sterile, ice-cold 0.1 M CaCl2 /15% glycerol (DO NOT VORTEX).
		+
		+	**Optional: Leave on ice 4 to 21 hours (BL21 for 4 hours, DHa and XL-1 blue for 0/n).
		+
		+	* Freeze aliquots of cells in sterile and labeled microcentrifuge tubes.

	==Notes==		==Notes==

Alex Zorychta: New page: ==Overview== This is how to isolate the phage genome from a preparation of bacteriophages, specifically the ''Bordetella'' phage. ==Materials== * 10 mM EDTA * CsCl-banded phage * steril...

2012-06-04T14:25:55Z

New page: ==Overview== This is how to isolate the phage genome from a preparation of bacteriophages, specifically the ''Bordetella'' phage. ==Materials== * 10 mM EDTA * CsCl-banded phage * steril...

New page

==Overview==

This is how to isolate the phage genome from a preparation of bacteriophages, specifically the ''Bordetella'' phage.

==Materials==

* 10 mM EDTA
* CsCl-banded phage
* sterile tube
* 100% ethanol
* -20°C freezer
* Centrifuge
* 0.5 mL of 70% ethanol
* TE Buffer

==Procedure==

* Add 10 volumes of 10mM EDTA pH 8.0 to 1 volume of CsCl-banded phage in a sterile tube
* Heat for 5 minutes at 65°C; let cool to room temperature
* Add 2.5 volumes 100% ethanol; place in -20°C freezer for 20 minutes
* Centrifuge: 10,000g for 5 minutes
* Discard supernatant; rinse pellet with 0.5ml 70% ethanol; centrifuge again; discard supernatant; rinse pellet with 0.5ml 70% ethanol; centrifuge again; discard supernatant
* Let pellet air dry at room temperature
* Resuspend DNA in TE buffer; let DNA sit for several hours to go into solution; or, restriction digests can be performed immediately; store solution of DNA at -20°C for long-term storage (i.e. months) or at 4°C for short-term storage (i.e. 1-2 days)

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



==References==


==Contact==
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[[Category:Protocol]]