Difference between revisions of "PMAD"

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(Created page with "250px|thumb|right|'''pGP1331: click to enlarge''' '''General comments''' Integrative plasmid for the fusion of 3x FLAG tag to the C-terminus of a prote...")
 
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[[File:PGP1331.png|250px|thumb|right|'''pGP1331: click to enlarge''']]
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[[File:pMAD.jpg|250px|thumb|right|'''pMAD: click to enlarge''']]
  
 
'''General comments'''
 
'''General comments'''
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'''Hints for the usage'''
 
'''Hints for the usage'''
* amplify the last 600bp of your gene of interest,
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* for a markerless deletion of a gene amplify 1000 bp up- and downstream of the gene and clone them into the MCS of the plasmid
* skip the Stopp-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 reference'''
 
'''The reference'''
<pubmed> 20572937</pubmed>
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<pubmed> 15528558 </pubmed>

Revision as of 09:03, 28 August 2012

pMAD: 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

  • for a markerless deletion of a gene amplify 1000 bp up- and downstream of the gene and clone them into the MCS of the plasmid

The reference

Maryvonne Arnaud, Arnaud Chastanet, Michel Débarbouillé
New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria.
Appl. Environ. Microbiol.: 2004, 70(11);6887-91
[PubMed:15528558] [WorldCat.org] [DOI] (P p)