Difference between revisions of "PGP1087"
| Line 9: | Line 9: | ||
* ''B. subtilis'': no ori, select for erythromycin/lincomycin | * ''B. subtilis'': no ori, select for erythromycin/lincomycin | ||
| − | * based pMUTIN-FLAG | + | * based on pMUTIN-FLAG |
'''Hints for the usage''' | '''Hints for the usage''' | ||
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 on 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
