Difference between revisions of "PGP1331"
(Created page with ' '''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.col…') |
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| + | [[File:PGP1331.png|250px|thumb|right|'''pGP1331: click to enlarge''']] | ||
| + | '''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 | |
| − | Integrative | + | * ''B. subtilis'': no ori, select for spectinomycin |
| − | E.coli: multicopy plasmid, select for ampicillin | ||
| − | B. subtilis: no ori, select for spectinomycin | ||
'''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)
