Paris/August 14

From 2007.igem.org

< Paris(Difference between revisions)
(MiniPrep and glycerol stock)
 
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[[Paris/August 13|yesterday]] -- [[Paris/August 15|tomorrow]]
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== Transformation of a lox-KmR-lox strain with a pBAD-CRE plasmid ==
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Francois Xavier Barre gave us the strain FX85 which as a lox-Km-lox in its chromosome. We will prepare CaCl2 competent cells of FX85 and transform it with a plasmid bearing pBAD-CRE. This will allow us measuring the excision frequency of the CRE/LOX system with different levels of induction
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'''Competent Cells:'''
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* Grow bacteria from fresh streak in 30ml LB until OD 0.6
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* Cool by swirling in ice water. pour in 50ml tube and centrifuge 3000rpm for 10min at 4°C
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* Pour off supernatant and resuspend in 20ml ice-cold CaCl2 0.1M. Sit on ice 20min.
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* Centrifuge as before and resuspend in 0.5ml of Cacl2 0.1M. Sit on ice.
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'''Transformation:'''
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* Mix 100µl of cells with 10µl DNA. Sit on ice 20min.
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* Heat shock 2min at 37°C. Put on ice 2min.
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* Add 2ml LB
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* Incubate 1H at 37°C
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* Plate 100µl and 1.5 ml
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==MiniPrep and glycerol stock ==
==MiniPrep and glycerol stock ==
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{|
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|- style="background: #ccccff;"
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! colspan="5" style="background: #ccccff;" | Plasmids: MiniPrep Products
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|- style="background: #ccccff; text-align: center;"
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|width=5%| Number
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|width=25%| Name
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|width=50%| Description
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|width=15%| Products in use
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|width=5%| Date
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |MP13
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|<bbpart>BBa_J61002</bbpart>
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|ptet-mRFP <partinfo>J61002 SpecifiedComponents</partinfo>
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|MP13.1, MP13.2
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| [[Paris/August 14| August 14]]
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|- style="background: #cccccc;" 
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| style="background: #ccffcc;" |MP14
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|<bbpart>BBa_J61025</bbpart>
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|Medium constitutive promoter in J61002 (clones 1 + 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo>
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|MP14.1, MP14.2
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| [[Paris/August 14| August 14]]
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|}
* <bbpart>J61002</bbpart>
* <bbpart>J61002</bbpart>
* <bbpart>J61025</bbpart>
* <bbpart>J61025</bbpart>
* <bbpart>B0015</bbpart>
* <bbpart>B0015</bbpart>

Latest revision as of 16:32, 16 August 2007

yesterday -- tomorrow

Transformation of a lox-KmR-lox strain with a pBAD-CRE plasmid

Francois Xavier Barre gave us the strain FX85 which as a lox-Km-lox in its chromosome. We will prepare CaCl2 competent cells of FX85 and transform it with a plasmid bearing pBAD-CRE. This will allow us measuring the excision frequency of the CRE/LOX system with different levels of induction

Competent Cells:

  • Grow bacteria from fresh streak in 30ml LB until OD 0.6
  • Cool by swirling in ice water. pour in 50ml tube and centrifuge 3000rpm for 10min at 4°C
  • Pour off supernatant and resuspend in 20ml ice-cold CaCl2 0.1M. Sit on ice 20min.
  • Centrifuge as before and resuspend in 0.5ml of Cacl2 0.1M. Sit on ice.

Transformation:

  • Mix 100µl of cells with 10µl DNA. Sit on ice 20min.
  • Heat shock 2min at 37°C. Put on ice 2min.
  • Add 2ml LB
  • Incubate 1H at 37°C
  • Plate 100µl and 1.5 ml

MiniPrep and glycerol stock

Plasmids: MiniPrep Products
Number Name Description Products in use Date
MP13 BBa_J61002 ptet-mRFP MP13.1, MP13.2 August 14
MP14 BBa_J61025 Medium constitutive promoter in J61002 (clones 1 + 2) MP14.1, MP14.2 August 14