Difference between revisions of "PGP1087"
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'''General comments''' | '''General comments''' | ||
| − | Integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping the control of the expression under its natural promotor | + | Integrative [[plasmid]] for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping the control of the expression under its natural promotor, allows express of the downstream gene by the p(spac) promoter |
| − | * ''E.coli'': | + | |
| + | * ''E.coli'': low copy plasmid, select for ampicillin | ||
* ''B. subtilis'': no ori, select for erythromycin/lincomycin | * ''B. subtilis'': no ori, select for erythromycin/lincomycin | ||
| + | |||
| + | * based pMUTIN-FLAG | ||
'''Hints for the usage''' | '''Hints for the usage''' | ||
* amplify the last 300bp of your gene of interest, | * amplify the last 300bp of your gene of interest, | ||
| − | * skip the | + | * skip the stop codon of the gene in the reverse primer |
* take care that the integration is in-frame | * take care that the integration is in-frame | ||
'''Antibody''' | '''Antibody''' | ||
*dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it) | *dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it) | ||
| + | |||
| + | constructed in the [[Stülke]] lab (C. Diethmaier) | ||
Revision as of 11:59, 17 March 2010
General comments
Integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a protein, keeping the control of the expression under its natural promotor, allows express of the downstream gene by the p(spac) promoter
- E.coli: low copy plasmid, select for ampicillin
- B. subtilis: no ori, select for erythromycin/lincomycin
- based pMUTIN-FLAG
Hints for the usage
- amplify the last 300bp of your gene of interest,
- skip the stop codon of the gene in the reverse primer
- take care that the integration is in-frame
Antibody
- dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it)
constructed in the Stülke lab (C. Diethmaier)
