Team:Macquarie Australia/Protocols/Making competent Cells

From 2012.igem.org

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

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:


  • 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
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