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Team:Macquarie Australia/Protocols/Making competent Cells
From 2012.igem.org
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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) | 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) | ||
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Experimental procedure: | Experimental procedure: | ||
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* Competent E. coli BL21(DE3) competent cells were obtained after growth and storage at -80C. | * Competent E. coli BL21(DE3) competent cells were obtained after growth and storage at -80C. | ||
* The tubes were defrosted in team mates hand. They were then put on ice | * The tubes were defrosted in team mates hand. They were then put on ice | ||
Revision as of 09:00, 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:
- Competent E. coli BL21(DE3) competent cells were obtained after growth and storage at -80C.
- The tubes were defrosted in team mates hand. They were then put on ice
- Add 1-10 µl of ligation mix to each tube. Incubated on ice for 5 min.
- Tubes were heat shocked at 42C in water bath for 30 seconds and returned to ice for 2 min.
- 200 µl of SOC media was added to each tube and incubated in the 37C shaker for 1 hour.
- 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.
- The plates were then placed in an incubator at 37C for growth overnight
