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Integrated Genomics icon

Integrated Genomics: A Discovery-Based Laboratory Course
(Kit # 1000000004 )

Depositing Labs:   Guy Caldwell

This vector kit contains 13 plasmids used in Integrated Genomics: A Discovery-Based Laboratory Course, by Guy Caldwell, Shelli Williams, and Kim Caldwell. These plasmids were deposited at Addgene by the Caldwell lab and the University of Alabama for distribution to academic and nonprofit research laboratories for educational use as part of the Integrated Genomics Discovery-Based Laboratory Course. Please note that the pACT2.2 C. elegans yeast 2-hybrid library derived from the original Caldwell library is not currently available from Addgene as supplies of the library have been exhausted.

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$400 USD + shipping

Available to academics and nonprofits only.

Original Publication

Integrated Genomics: A Discovery-Based Laboratory Course. Caldwell GA, Williams SN, Caldwell KA John Wiley and Sons 2006. Textbook .

Description

The discovery-based genomics course developed by the Caldwell laboratory includes experiments using the plasmids available in this kit with C. elegans as a teaching resource. These plasmids may not be suitable for research purposes.

Protocols

Detailed experimental protocols for use in the Integrated Genomics course can be found in the Integrated Genomics: A Discovery-Based Laboratory Course textbook .

Links

How to Cite this Kit

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.

For your Materials and Methods section:
"The C. elegans kit used with the Integrated Genomics course was a gift from Guy Caldwell (Addgene kit # 1000000004)"

This kit consists of the following plasmids:

  • GFP::L4440- Full-length GFP sequence (from vectorp95_77, gift of Dr. Andrew Fire) cloned into RNAi feeding vectorL4440. Use in a strain carrying a GFP construct to observe reduction of GFP signal after RNAi induction as outlined in Chapter 8.

  • lis-1::L4440- Full length cDNA for C. elegans open-reading frameT03F6.5, encoding thelis-1 gene, cloned into RNAi feeding vectorL4440. Can be used for RNAi feeding; should result in embryonic lethality at high levels of induction as outlined in Chapter 8.

  • nud-1::L4440- Full length cDNA for C. elegans open-reading frameF53A2.4, encoding thenud-1 gene, cloned into RNAi feeding vectorL4440. Can be used for RNAi feeding; should result in embryonic lethality at high levels of induction as outlined in Chapter 8.

  • pLexA::lis-1- Full-length cDNA for C. elegans open-reading frameT03F6.5, encoding thelis-1 gene, cloned into the yeast two-hybrid DNA-binding domain vectorpLexA(between EcoRI and PstI sites). This vector can be used as the bait for a yeast two-hybrid screen as outlined in Chapters 3 and 4.

  • pLexA::nud-1- Full-length cDNA for C. elegans open-reading frameF53A2.4, encoding thenud-1 gene, cloned into the yeast two-hybrid DNA-binding domain vectorpLexA(between EcoRI and BamHI sites). This vector can be used as the bait for a yeast two-hybrid screen as outlined in Chapters 3 and 4.

  • pLexA::Ras- cDNA of fission yeast S. pombeRas1gene (oncogene) cloned into the yeast two-hybrid DNA-binding domain vectorpLexA(ref: Chang et al., 2004, Cell, 79:131-141,PubMed). This vector is used as a positive control in the yeast two-hybrid screen outlined in Chapters 3 and 4.

  • pAct::Raf- cDNA of fission yeast S. pombebyr-2gene (oncogene homolog) cloned into the yeast two-hybrid activation-domain vector (also called, pGADGH-byr2, Van Aelst et al., 1993, PNAS, 90(13):6213-7,PubMed). This vector is used as a positive and negative control in the yeast two-hybrid experiments outlined in Chapters 3 and 4.

  • L4440- RNAi feeding vector, containing the MCS illustrated in Figure 7.1. Use for traditional cloning of an RNAi target sequence for RNAi feeding experiments.

  • pLexA- Yeast two-hybrid DNA-binding domain vector, containing the MCS illustrated in Figure A. Use for traditional cloning for a target cDNA sequence for a yeast two-hybrid screen.

  • pACT2.2- Yeast two-hybrid transcriptional activation domain vector, containing the MCS illustrated in Figure 4.1. Use for traditional cloning for a target cDNA sequence to test a directed yeast two-hybrid interaction (no screening involved).

  • L4440gtwy- Gateway-modified RNAi feeding vector. Use this vector for Gateway cloning of an RNAi target sequence as outlined in Alternative Chapter 7. A Gateway recombinational cassette was placed into the EcoRV site of vectorL4440to generate this modified plasmid. To facilitate subcloning of a given DNA insert into this plasmid, Gateway recombinational attachment sites should be incorporated into primers used for amplificiation, as outlined in the Invitrogen Gateway manual. This vector is propagated in E. coli strain DB3.1, due to the requirement to circumvent the lethality of the inherent ccdB gene until an insert is cloned into the MCS.

  • pLexAgtwy- Gateway-modified yeast two-hybrid DNA-binding domain vector. Use this vector for Gateway cloning of a cDNA to be used as the bait in a yeast two-hybrid screen. Gateway cloning is outlined in Alternative Chapter 7. This vector was modified from plasmidpLexAby placing a Gateway acceptor cassette in the SmaI site of pLexA. To facilitate subcloning of a given DNA insert into this plasmid, Gateway recombinational attachment sites should be incorporated into primers used for amplificiation, as outlined in the Invitrogen Gateway manual. Gateway recombinational cassettes should be added to primers as outlined in the Invitrogen Gateway manual. This vector is propagated in E. coli strain DB3.1, due to the requirement to circumvent the lethality of the inherent ccdB gene until an insert is recombined into the Gateway cassette.

  • pACT2.2gtwy- Gateway-modified yeast two-hybrid Activation domain vector. Use this vector for Gateway cloning of a cDNA to be used to test a directed yeast two-hybrid interaction (no screening involved). Gateway cloning is outlined in Alternative Chapter 7. This vector was modified from plasmid pACT2.2 by placing a Gateway acceptor cassette in the SmaI site ofpACT2.2. To facilitate subcloning of a given DNA insert into this plasmid, Gateway recombinational attachment sites should be incorporated into primers used for amplificiation, as outlined in the Invitrogen Gateway manual. This vector is propagated in E. coli strain DB3.1, due to the requirement to circumvent the lethality of the inherent ccdB gene until an insert is recombined into the Gateway cassette.

Not included in this kit:

  • pACT2.2 C. elegans yeast 2-hybrid library- The C. elegans yeast two-hybrid cDNA library was constructed by Lindsay Faircloth in the Caldwell Laboratory 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, Gateway™ Technology is utilized to recombine the cDNA products into the entry vector pDONR222. From this entry library, Gateway-mediated recombination allows for introduction into any number of expression vectors. In the case of this two-hybrid library, entry clones were recombined intopACT2.2gtwy. Large preparations of purified library were prepared using Invitrogen's S.N.A.P. Midiprep kit for nucleic acid purification. Please note that supplies of this library have been exhausted and it is discontinued.

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