Paris/September 18
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| + | == DapA Wanner deletion: pKD13-DapA homologies PCR == | ||
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| + | We diluted 1/100 overnight culture at 30°C of MG1655 transformed by pKD46: 50µL into 5mL LB ampicilline.<br> | ||
| + | At OD = 0.1, we induced pKD46 expression by addition of arabinose (10mM final). <br> | ||
| + | At OD = 0.4, we prepared cells to be electrocompetent. (see [[Paris/PROTOCOLS#Recombineering.2FLambda_red-mediated_gene_replacement|Protocol]])<br> | ||
| + | Then we transformed the bacteria with PCR product from yesterday (see [[Paris/September_18|September 18]]). | ||
| + | |||
== Minipreps == | == Minipreps == | ||
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*S26: (lRBS-DapAsubtilis) MPT5.? (préciser David) | *S26: (lRBS-DapAsubtilis) MPT5.? (préciser David) | ||
*pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé | *pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé | ||
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== Digestion reactions == | == Digestion reactions == | ||
Revision as of 09:32, 20 September 2007
Contents |
DapA Wanner deletion: pKD13-DapA homologies PCR
We diluted 1/100 overnight culture at 30°C of MG1655 transformed by pKD46: 50µL into 5mL LB ampicilline.
At OD = 0.1, we induced pKD46 expression by addition of arabinose (10mM final).
At OD = 0.4, we prepared cells to be electrocompetent. (see Protocol)
Then we transformed the bacteria with PCR product from yesterday (see September 18).
Minipreps
Minipreps sur cultures ON de:
- S24: (lox66-DapAsubtilis) MPT3.? (préciser David)
- S26: (lRBS-DapAsubtilis) MPT5.? (préciser David)
- pKD13: (plasmide FRT-KanR-FRT de Wanner), un stock glycerol est également réalisé
Digestion reactions
| Digestion Products | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Number | Product Name | Matrix Name | Enzyme 1 | Enzyme 2 | Size | Description | |||
| D77 | araC/pBad>>lox71-gfptripart BI | D60 (.1 & .2) digestion product | FspI | AhdI | in D60, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion | ||||
| D78 | araC/pBad>>lox71-gfptripart FI | D71 (.1 & .2) digestion product | FspI | AhdI | in D71, the extracted fragment & the open plasmid have the same size, it is thus necessary to remove the plasmid by digestion | ||||
| D53 | RBS-dapAsubtilis BBinsert | pTOPO of RBS-dapAsubtilis (MPT5.2) | EcoRI | Pst1 | RBS-dapAsubtilis extracted for cloning as a biobrick | ||||
| D54 | RBS-dapAsubtilis BI | pTOPO of RBS-dapAsubtilis (MPT5.2) | XbaI | Pst1 | RBS-dapAsubtilis extracted for use as a BI | ||||
Ligation reactions
| Ligations | ||||||
|---|---|---|---|---|---|---|
| Number | Insert | Insert Volume (µL) | Vector | Vector Volume (µL) | Comments | Number of colonies |
| C1 | MP3.1 digested with EcoRI (D0) | 2 | control of ligation efficiency | |||
