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-	==Biobricks==	+	==Competent cell protocol==
-		+
		+	[[File:nitrogen.jpg|500px|thumb|left|Competent cells were reduced to -80C via liquid nitrogen]]
	====Methods:====		====Methods:====
-	Transformation of E. coli competent cells	+	<p>
-	Experimental procedure:	+	In order to produce competent cells for our transformations, we used a modified version of the protocol developed by Inoue et al (1990).
-		+	</p>
-		+	<p>
-	1) Obtain competent E coli cells from -80 C.	+	We grew cultures of E. coli cells (Top10 and BL21 strains) overnight with shaking at room temperature in SOB media, to mid-log phase to an A600 of ~0.5. They were then chilled on ice and pelleted in the Sigma centrifuge at ~2500g  for about seven minutes at 4°C. We then discarded the supernatant and placed the cells back on ice, before resuspending them in 1 to 5mL of ice-cold TB buffer and using additional buffer to bring the volume up to about one-fifth of the original volume. This solution was incubated on ice for ten minutes, before we centrifuged it again and discarded the supernatant. Again, we resuspended the cells in about one-twentieth of the original culture volume of ice-cold TB buffer. We then distributed 930µL of this cell solution into a number of 1.5mL microcentrifuge tubes (that had been pre-chilled on ice) and added 70µL of DMSO to each. Each tube was gently mixed and placed on ice. These were then divided into smaller aliquots (100µL) and frozen for later use.
-	2) Hold the tubes of competent cells in your fingers until they are just defrosted, and then put them on ice immediately. 100 µl is sufficient for 2 transformations.	+	</p>
-	3) Add 1-10 µl of ligation mix to each tube. Incubate on ice for 5 min.	+	<p>
-	4) Put the tubes in the 42°C bath for 30 sec and put back on ice for 2 min.	+	Details of the original protocol are located in Inoue et al. (1990) "High efficiency transformation of Escherichia coli with plasmids", ''Gene'', vol. 96, pp. 23-38.
-	5) Add 200 µl of SOC media to each tube, and incubate in the 37oC shaker for 10 min or up to 1hr. I do 10 minutes if it is just a plasmid or 1 hour for ligation mixes.	+
-	6) For each tube of cells, spread 5 µl onto one LB (Ampicillin) plate and 50 µl onto a second LB (Ampicillin) plate, using aseptic technique. This should give you a total of four plates.	+
-	7) Place your plates upside-down in the 37°C incubator.	+

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Latest revision as of 00:33, 4 September 2012

Competent cell protocol

Methods:

In order to produce competent cells for our transformations, we used a modified version of the protocol developed by Inoue et al (1990).

We grew cultures of E. coli cells (Top10 and BL21 strains) overnight with shaking at room temperature in SOB media, to mid-log phase to an A600 of ~0.5. They were then chilled on ice and pelleted in the Sigma centrifuge at ~2500g for about seven minutes at 4°C. We then discarded the supernatant and placed the cells back on ice, before resuspending them in 1 to 5mL of ice-cold TB buffer and using additional buffer to bring the volume up to about one-fifth of the original volume. This solution was incubated on ice for ten minutes, before we centrifuged it again and discarded the supernatant. Again, we resuspended the cells in about one-twentieth of the original culture volume of ice-cold TB buffer. We then distributed 930µL of this cell solution into a number of 1.5mL microcentrifuge tubes (that had been pre-chilled on ice) and added 70µL of DMSO to each. Each tube was gently mixed and placed on ice. These were then divided into smaller aliquots (100µL) and frozen for later use.

Details of the original protocol are located in Inoue et al. (1990) "High efficiency transformation of Escherichia coli with plasmids", Gene, vol. 96, pp. 23-38.