Paris/August 14
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| + | [[Paris/August 13|yesterday]] -- [[Paris/August 15|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 == | ==MiniPrep and glycerol stock == | ||
| + | {| | ||
| + | |- style="background: #ccccff;" | ||
| + | ! colspan="5" style="background: #ccccff;" | Plasmids: MiniPrep Products | ||
| + | |- style="background: #ccccff; text-align: center;" | ||
| + | |width=5%| Number | ||
| + | |width=25%| Name | ||
| + | |width=50%| Description | ||
| + | |width=15%| Products in use | ||
| + | |width=5%| Date | ||
| + | |- style="background: #cccccc;" | ||
| + | | style="background: #ccffcc;" |MP13 | ||
| + | |<bbpart>BBa_J61002</bbpart> | ||
| + | |ptet-mRFP <partinfo>J61002 SpecifiedComponents</partinfo> | ||
| + | |MP13.1, MP13.2 | ||
| + | | [[Paris/August 14| August 14]] | ||
| + | |- style="background: #cccccc;" | ||
| + | | style="background: #ccffcc;" |MP14 | ||
| + | |<bbpart>BBa_J61025</bbpart> | ||
| + | |Medium constitutive promoter in J61002 (clones 1 + 2) <partinfo>BBa_J61025 SpecifiedComponents</partinfo> | ||
| + | |MP14.1, MP14.2 | ||
| + | | [[Paris/August 14| August 14]] | ||
| + | |} | ||
* <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
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 |
