Multisite Gateway Cloning for C. elegans

Molecular biology was performed according to standard protocols. The Multisite Gateway Three-Fragment Vector Construction Kit (Life Technologies, Grand Island, NY) was used to construct donor plasmids. Fragments were then recombined into the MosSCI destination vectors pCFJ150 or pCFJ212 (Frøkjær-Jensen et al., 2008 (link), 2012 (link)). For constructs in which two fragments were ligated by PCR, fusion PCR was performed as in Hobert (2002) (link). Primers used for generating constructs are listed in Table 3, and plasmid construction strategies are summarized in Table 4.

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Hansen J.M., Chavez D.R, & Stanfield G.M. (2015). COMP-1 promotes competitive advantage of nematode sperm. eLife, 4, e05423.

Publication 2015

Multisite Gateway Three-Fragment Vector Construction Kit

Donor Plasmid Primers Vectors

Corresponding Organization :

Other organizations : University of Utah

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Variable analysis

independent variables

The use of the Multisite Gateway Three-Fragment Vector Construction Kit (Life Technologies, Grand Island, NY) to construct donor plasmids
The use of the MosSCI destination vectors pCFJ150 or pCFJ212 (Frøkjær-Jensen et al., 2008, 2012) for recombination
The use of fusion PCR to ligate two fragments, as described in Hobert (2002)

dependent variables

The resulting constructs, as described in Tables 3 and 4

control variables

Standard molecular biology protocols were followed

controls

No positive or negative controls were explicitly mentioned in the input protocol

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