Insertion Libraries

Three insertion libraries are available. All are derived by using mini-transposons (mTns) to mutagenize a yeast genomic DNA library constructed in the vector pHSS6 by Brian Grimwade. See Genes and Dev. 8:1087-1105, 1994 for a description of how the genomic library was made.

• Library with insertions of mTn-lacZ/LEU2
• Library with insertions of mTn-3xHA/lacZ
• Library with insertions of mTn-3xHA/GFP

Library with insertions of mTn-lacZ/LEU2

This insertion library was used for the pilot project (Genes and Dev. 8:1087-1105, 1994). The mTn-lacZ/LEU2 transposon used for mutagenesis is originally described in Siefert et al. (1986).

mTn contains:

• 5'-truncated lacZ gene encoding beta-galactosidase
• LEU2
• Amp resistance

Suggested uses:

• Insertional mutagenesis of the yeast genome.
• Analysis of differential gene expression.
• Identification of downstream targets.
• Immunodetection of fusion protein.

Rescue methods:

• pRSQ2-URA3
• pRSQ
• YIp5
• vectorette PCR

More information:

• Outline of how the library is used
• Detailed protocol
• Request library

Library with insertions of mTn-3xHA/lacZ

This insertion libray is being used in our current project. The mTn-3xHA/lacZ transposon used for mutagenesis was constructed and tested by Amy Sheehan and Petra Ross-Macdonald in this laboratory.

mTn contains:

• 5'-truncated lacZ gene encoding beta-galactosidase
• Hemagglutinin (HA) triple epitope tag
• URA3
• Tet resistance

Suggested uses:

• Insertional mutagenesis of the yeast genome.
• Analysis of differential gene expression via assays for beta-gal activity.
• Obtaining epitope-tagged protein from mutagenized gene.
• Immunodetection of fusion protein or epitope tagged protein.

Rescue methods:

• pRSQ2-LEU2
• This vectorette PCR protocol has been adapted for use with mTn-3xHA/lacZ, but not tested.

More information:

• Outline of how the library is used
• Detailed protocol
• Request library

Library with insertions of mTn-3xHA/GFP

This insertion libray has not been used by us. The mTn-3xHA/GFP transposon used for mutagenesis was constructed and tested by Amy Sheehan and Petra Ross-Macdonald in this laboratory.

mTn contains:

• coding region for GFP mutant p11
• Hemagglutinin (HA) triple epitope tag
• URA3
• Tet resistance gene

Suggested uses:

• Insertional mutagenesis of the yeast genome.
• Analysis of gene expression.
• Obtaining epitope-tagged protein from mutagenized gene.
• Fluorodetection of GFP fusion protein.
• Immunodetection of epitope tagged protein.

Rescue method:

• No vector has been constructed.
• Inverse PCR could be used.
• This vectorette PCR protocol has been adapted for use with mTn-3xHA/GFP, but not tested.

More information:

• Outline of how the library is used
• Detailed protocol
• Request library