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, allows express of the downstream gene by the p(spac) promoter | + | * 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 |
| + | * constructed in [[Jörg Stülke]]'s lab (C. Diethmaier) | ||
* ''E.coli'': low copy plasmid, select for ampicillin | * ''E.coli'': low copy plasmid, select for ampicillin | ||
| Line 18: | Line 19: | ||
*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) | ||
| − | + | =The references= | |
| + | <big>''Diethmaier C, Pietack N, Gunka K, Wrede C, Lehnik-Habrink M, Herzberg C, Hübner S, Stülke J'' </big> | ||
| + | <big>'''A Novel Factor Controlling Bistability in ''Bacillus subtilis'': The YmdB Protein Affects</big> | ||
| + | <big>Flagellin Expression and Biofilm Formation.''' </big> | ||
| + | J Bacteriol.: 2011, 193(21):5997-6007. | ||
| + | [http://www.ncbi.nlm.nih.gov/pubmed/21856853 PubMed:21856853] | ||
Revision as of 15:31, 31 October 2011
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
- constructed in Jörg Stülke's lab (C. Diethmaier)
- 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)
The references
Diethmaier C, Pietack N, Gunka K, Wrede C, Lehnik-Habrink M, Herzberg C, Hübner S, Stülke J A Novel Factor Controlling Bistability in Bacillus subtilis: The YmdB Protein Affects Flagellin Expression and Biofilm Formation. J Bacteriol.: 2011, 193(21):5997-6007. PubMed:21856853
