Difference between revisions of "PGP1331"
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'''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! | * Don't use XbaI and KpnI for cloning. They cut the vector twice! | ||
Revision as of 11:55, 7 December 2012
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)
