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KG#81
(Plasmid #110876)

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 110876 Standard format: Plasmid sent in bacteria as agar stab 1 $85

This material is available to academics and nonprofits only.

Backbone

  • Vector backbone
    pUC
  • Backbone size w/o insert (bp) 2617
  • Total vector size (bp) 9853
  • Vector type
    Bacterial Expression

Growth in Bacteria

  • Bacterial Resistance(s)
    Ampicillin, 100 μg/mL
  • Growth Temperature
    37°C
  • Growth Strain(s)
    DH5alpha
  • Growth instructions
    Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).
  • Copy number
    High Copy

Gene/Insert 1

  • Gene/Insert name
    myo-3 promoter
  • Species
    C. elegans (nematode)
  • Insert Size (bp)
    2387

Cloning Information for Gene/Insert 1

Gene/Insert 2

  • Gene/Insert name
    acy-1 P260S gain-of-function cDNA
  • Species
    C. elegans (nematode)
  • Insert Size (bp)
    3762
  • Mutation
    Changed Proline to Serine at amino acid 260

Cloning Information for Gene/Insert 2

Gene/Insert 3

  • Gene/Insert name
    unc-54 3' control region with 1 artificial intron just upstream and 1 artificial intron in control region
  • Species
    C. elegans (nematode)
  • Insert Size (bp)
    978

Cloning Information for Gene/Insert 3

Resource Information

Terms and Licenses

  • Academic/Nonprofit Terms
  • Industry Terms
    • Not Available to Industry
Trademarks:
  • Zeocin® is an InvivoGen trademark.

Depositor Comments

Always streak out this culture for single colonies and do minipreps on >= 4 clones (~1/2 of colonies will be right). Choose small colonies after 18 - 20h growth. DNA yeild is low (~170 ng/ ul x 30 ul for a Qiagen 3 ml miniprep).

Used Pfu Ultra polymerase and primers engineered with restriction sites to amplify the 3.8 Kb acy-1(P260S) cDNA coding region from KG#63 and cloned into Age I/ Xho I cut pPD96.52 (myo-3:: expression vector). Transformed into XL1-Blue electrocompetent cells. Miniprepped 8 clones to find those with correct size insert and vector, then submitted 2 clones for sequence. Chose one clone that has correct sequence and make glycerol stock and save the DNA.

Features of the expression construct: This expression construct has a promoter (myo-3) that will drive strong expression of acy-1 (P260S) cDNA in the body wall muscle cells. The myo-3 promoter is generally active in body muscles (body wall, vulval, and intestine associated muscles including the anal depressor [Okkema et al., 1993 Genetics; Ardizzi and Epstein 1987 JCB]. Other features of the expression vector include the unc-54 3' end including the poly A addition signal, and 3 introns (1 in 5' UTR, 1 just upstream of the unc-54 3' end, and 1 inserted in the unc-54 3' end). These introns help provide more uniform and robust expression, especially if you are expressing a cDNA with no introns (as we are) as opposed to a gene with introns. The sequence AAAA is engineered just after the 5' restriction site and just before the initiator ATG to provide a consensus translation start site. The sequence AT follows the stop codon and immediately precedes the 3' restriction site to provide a consensus post - stop codon sequence. Note: the acy-1 cDNA from which this clone was produced corresponds to the single acy-1 cDNA predicted by wormbase, which is F17C8.1 except for the gf mutation in this clone.

How to cite this plasmid ( Back to top)

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

  • For your Materials & Methods section:

    KG#81 was a gift from Kenneth Miller (Addgene plasmid # 110876 ; http://n2t.net/addgene:110876 ; RRID:Addgene_110876)
  • For your References section:

    Mutations that rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the G alpha(s) pathway and define a third major branch of the synaptic signaling network. Schade MA, Reynolds NK, Dollins CM, Miller KG. Genetics. 2005 Feb;169(2):631-49. doi: 10.1534/genetics.104.032334. Epub 2004 Oct 16. 10.1534/genetics.104.032334 PubMed 15489510