Paris/August 14
From 2007.igem.org
(Difference between revisions)
(→Transformation of a lox-KmR-lox strain with a pBAD-CRE plasmid) |
(→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 | 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 | * 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 | * Cool by swirling in ice water. pour in 50ml tube and centrifuge 3000rpm for 10min at 4°C | ||
| Line 8: | Line 9: | ||
* Centrifuge as before and resuspend in 0.5ml of Cacl2 0.1M. Sit on ice. | * Centrifuge as before and resuspend in 0.5ml of Cacl2 0.1M. Sit on ice. | ||
| - | Transformation: | + | '''Transformation:''' |
* Mix 100µl of cells with 10µl DNA. Sit on ice 20min. | * Mix 100µl of cells with 10µl DNA. Sit on ice 20min. | ||
* Heat shock 2min at 37°C. Put on ice 2min. | * Heat shock 2min at 37°C. Put on ice 2min. | ||
Revision as of 11:07, 14 August 2007
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
