Difference between revisions of "PGP1331"
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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 |
* ''E.coli'': multicopy plasmid, select for ampicillin | * ''E.coli'': multicopy plasmid, select for ampicillin | ||
* ''B. subtilis'': no ori, select for spectinomycin | * ''B. subtilis'': no ori, select for spectinomycin | ||
| Line 9: | Line 9: | ||
'''Hints for the usage''' | '''Hints for the usage''' | ||
* amplify the last 600bp of your gene of interest, | * amplify the last 600bp 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 | ||
| + | * Don't use XbaI and KpnI for cloning. They cut the vector twice! | ||
'''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) | ||
| + | |||
| + | '''The reference''' | ||
| + | <pubmed> 20572937</pubmed> | ||
| + | == Return to [[Plasmids]] == | ||
Latest revision as of 13:58, 3 June 2013
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
- E.coli: multicopy plasmid, select for ampicillin
- B. subtilis: no ori, select for spectinomycin
Hints for the usage
- amplify the last 600bp of your gene of interest,
- skip the Stop-codon of the gene in the reverse primer
- take care that the integration is in-frame
- Don't use XbaI and KpnI for cloning. They cut the vector twice!
Antibody
- dilute the anitbody 1:10000 (you can freeze the Blotto solution and reuse it)
The reference
Martin Lehnik-Habrink, Henrike Pförtner, Leonie Rempeters, Nico Pietack, Christina Herzberg, Jörg Stülke
The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complex.
Mol Microbiol: 2010, 77(4);958-71
[PubMed:20572937]
[WorldCat.org]
[DOI]
(I p)
