|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||47947||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Vector backbonepBluescript SK+
- Backbone size w/o insert (bp) 2958
- Total vector size (bp) 8127
Vector typeWorm Expression, CRISPR
Growth in Bacteria
Growth instructionsPlasmid isolation from standard DH5alpha results in low yield. The plasmid may need to first be retransformed into BL21 cells to obtain normal DNA yields. Requires extra growth time.
Insert Size (bp)4254
- Promoter hsp-16.48
/ Fusion Proteins
- 3xFLAG (N terminal on insert)
- SV40 NLS (N terminal on insert)
- egl-13 NLS (C terminal on insert)
- Cloning method Unknown
- 5′ sequencing primer M13F
- 3′ sequencing primer M13R (Common Sequencing Primers)
To generate the unique SpeI site, a short, 37 bp, linker was inserted into an existing NdeI site. This linker seems to have been inserted 2 or more times, creating a short repeat region that we have been unable to sequence across. Sequencing with the M13 reverse primer will fail.
This plasmid is provided in EPI400 bacteria which may need to be induced to obtain a high copy number. Please refer to this protocol: http://www.epibio.com/docs/default-source/protocols/copycutter-epi400-electrocompetent-e-coli-copycutter-epi400-chemically-competent-e-coli-copycutter-induction-solution.pdf?sfvrsn=10 for more information.
For more information on Boxem Lab CRISPR Plasmids please refer to: http://www.addgene.org/crispr/boxem/
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:pMB67 was a gift from Mike Boxem (Addgene plasmid # 47947 ; http://n2t.net/addgene:47947 ; RRID:Addgene_47947)
For your References section:CRISPR/Cas9-Targeted Mutagenesis in Caenorhabditis elegans. Waaijers S, Portegijs V, Kerver J, Lemmens BB, Tijsterman M, van den Heuvel S, Boxem M. Genetics. 2013 Aug 26. 10.1534/genetics.113.156299 PubMed 23979586