pACT2.2 C. elegans yeast 2-hybrid library
(Pooled library #11523)

• Depositing Lab: Guy Caldwell
• Publication: Integrated Genomics (unpublished)

Backbone

• Vector backbone: pACT2.2gtwy
• Backbone manufacturer: see Addgene plasmid 11346 for more information
• Backbone size w/o insert (bp): 7550
• Vector type: Yeast Expression
• Selectable markers: LEU2

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: C. elegans cDNA library
• Species: C. elegans (nematode)
• Tag / Fusion Protein:
  • transcriptional activation domain (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: Gal4 AD

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

A C. elegans yeast two-hybrid cDNA library was constructed using Invitrogen's CloneMiner cDNA Library Construction Kit. Use of this kit allows for the construction of highly representative libraries without the use of restriction enzyme cloning methods. Instead cDNA amplification from an mRNA template is performed using the SuperScript II reverse transcriptase (RT), generating high cDNA yields through reduction of RNA degradation during first strand synthesis. Following this amplification of large, often full-length genes, GatewayTM Technology is utilized to recombine the cDNA products into the entry vector pDONR222. From this entry library, GatewayTM -mediated recombination allows for introduction into any number of expression vectors. In the case of this two-hybrid library, entry clones were recombined into the Gateway-converted pACT2.2 vector (see plasmid 11346). Large preparations of purified library were prepared using Qiagen's Maxiprep kit for nucleic acid purification.

The library was subsequently tested for quality assurance. Over 90% of clones contained inserts. A random PCR amplification screen was then perform to test for full-length amplification of products, using primers to over 10 target genes, ranging in size from 800 to 2500bp. All PCR results confirmed the amplification of full-length cDNA products as visualized by agarose gel electrophoresis. No non-specific amplification of the Gateway cassette was observed using primers containing the Gateway cassette flanking a gene product not encoded within the C. elegans genome.

The library being distributed by Addgene is derived from the original Caldwell library. This library is intended to be used as part of the Integrated Genomics laboratory course. Addgene does not make any guarantee that the library includes complete representation of C. elegans cDNAs. Addgene will distribute the library in aliquots of 150 uL of 1 ug/uL DNA.