Difference between revisions of "PGP1331"

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* skip the Stopp-Codon of the gene in the reverse primer
 
* skip the Stopp-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'''

Revision as of 12:54, 7 December 2012

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
  • B. subtilis: no ori, select for spectinomycin

Hints for the usage

  • amplify the last 600bp of your gene of interest,
  • skip the Stopp-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)