Team:Macquarie Australia/Protocols/Making competent Cells

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Revision as of 08:58, 15 August 2012

Biobricks

Methods:

Transformation of E. coli competent cells (from Inoue et al., 1990 'High efficiency transformation of Escherichia coli with plasmids', Gene, vol. 96, pp. 23-28) Experimental procedure:

1)Competent E. coli BL21(DE3) competent cells were obtained after growth and storage at -80C.
2)The tubes were defrosted in team mates hand. They were then put on ice
3)Add 1-10 µl of ligation mix to each tube. Incubated on ice for 5 min.
4)Tubes were heat shocked at 42C in water bath for 30 seconds and returned to ice for 2 min.
5) 200 µl of SOC media was added to each tube and incubated in the 37C shaker for 1 hour.
6) Each tube of cells had 5 µl spread onto an LB (Ampicillin) plate and another 50 µl onto a second LB (Ampicillin) plate, using aseptic technique.
7) The plates were then placed in an incubator at 37C for growth overnight

@@ Line 7: / Line 7: @@
 ====Methods:====
-Transformation of E. coli competent cells
+Transformation of E. coli competent cells (from Inoue et al., 1990 'High efficiency transformation of Escherichia coli with plasmids', Gene, vol. 96, pp. 23-28)
 Experimental procedure:
-*1)	Obtain competent E coli cells from -80 C.
+*1)Competent E. coli BL21(DE3) competent cells were obtained after growth and storage at -80C.
-*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.
+*2)The tubes were defrosted in team mates hand. They were then put on ice
-*3)	Add 1-10 µl of ligation mix to each tube. Incubate on ice for 5 min.
+*3)Add '''1-10 µl''' of ligation mix to each tube. Incubated on ice for 5 min.
-*4)	Put the tubes in the 42°C bath for 30 sec and put back on ice for 2 min.
+*4)Tubes were heat shocked at 42C in water bath for 30 seconds and returned to ice for 2 min.
-*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.
+*5) 200 µl of SOC media was added to each tube and incubated in the 37C shaker for 1 hour.
-*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.
+*6) Each tube of cells had 5 µl spread onto an LB (Ampicillin) plate and another 50 µl onto a second LB (Ampicillin) plate, using aseptic technique.
-*7)	Place your plates upside-down in the 37°C incubator.
+*7) The plates were then placed in an incubator at 37C for growth overnight