This vector kit contains 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 non-profit research laboratories for research and educational use.

 
Price and Distribution:

Individual plasmids from this kit can be ordered at $65 per plasmid, except for L4440, which is $40 per plasmid. The pACT2.2 C. elegans yeast 2-hybrid 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. The price of the library is $300. Addgene will distribute the library in aliquots of 150 uL of 1ug/uL DNA. To order these items, please click on the Order buttons below.

Prices for the kits are:

Full Kit (13 plasmids and yeast 2-hybrid library	$700
Kit without the library (13 plasmids)	$400

Plasmids are shipped as bacterial stabs. The yeast 2-hybrid library is shipped as DNA.

Yeast strains and worm strains for this book are available from other repositories.

 
Plasmid Description:

GFP::L4440

Full-length GFP sequence (from vector p95_77, gift of Dr. Andrew Fire) cloned into RNAi feeding vector L4440. 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 frame T03F6.5, encoding the lis-1 gene, cloned into RNAi feeding vector L4440. 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 frame F53A2.4, encoding the nud-1 gene, cloned into RNAi feeding vector L4440. 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 frame T03F6.5, encoding the lis-1 gene, cloned into the yeast two-hybrid DNA-binding domain vector pLexA (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 frame F53A2.4, encoding the nud-1 gene, cloned into the yeast two-hybrid DNA-binding domain vector pLexA (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. pombe Ras1 gene (oncogene) cloned into the yeast two-hybrid DNA-binding domain vector pLexA (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. pombe byr-2 gene (Raf 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 L4440. 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 vector L4440 to 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 plasmid pLexA by 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 of pACT2.2. 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.

pACT2.2 C. elegans yeast 2-hybrid library

A 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 into pACT2.2gtwy. Large preparations of purified library were prepared using Invitrogen's S.N.A.P. Midiprep kit for nucleic acid purification.

This library is intended to be used as part of the Integrated Genomics laboratory course rather than for research. Addgene does not make any guarantee that the library includes complete representation of C. elegans cDNAs.

 
Other Materials For The Book:

S. cerevisiae strain L40 is available from ATCC, the ATCC number is MYA-3332.

Worm strains can be obtained from the Caenorhabditis Genetics Center (CGC).