Cloning Promoters into pGreenII 0800-LUC

A 0.97 kb region of the Arabidopsis CHS promoter (At5g13930) was amplified by PCR from the Arabidopsis ecotype Columbia using the primers RPH-179 and RPH-180, then digested with KpnI and NcoI and cloned into the MCS of pGreenII 0800-LUC. The 1.04 kb pea CHS-1a promoter [GenBank: X80007] was subcloned into pGreenII 0800-LUC as an EcoRI-NcoI fragment [28 ]. A 0.92 kb Petunia CHS-A promoter [GenBank: X14591] was amplified by PCR using primers RPH-332 and RPH-333 from a V26 genomic DNA, digested with KpnI and NcoI and cloned into pGreenII 0800-LUC. The 1.3 kb Apple CHS1 promoter [GenBank: DQ022678] was isolated from Malus domestica Royal Gala, using the Genome Walker kit (Clonetech) with gene specific primers RPH-198 and RPH-199, then cloned into pGEM T-easy (Promega) and subcloned as a SalI-NcoI fragment into pGreenII 0800-LUC. pwo-Polymerase (Roche) was used for all PCR amplifications and cloned genes were sequenced to confirm no sequence modifications were incorporated.

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Hellens R.P., Allan A.C., Friel E.N., Bolitho K., Grafton K., Templeton M.D., Karunairetnam S., Gleave A.P, & Laing W.A. (2005). Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods, 1, 13.

Publication 2005

Arabidopsis Ecori Ecotype Gala Genes Genome Malus domestica Petunia Pgem Primers Promega Pwo polymerase Walker

Corresponding Organization :

Other organizations : AgResearch

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Protocol cited in 36 other protocols

Variable analysis

independent variables

Arabidopsis CHS promoter (At5g13930)
Pea CHS-1a promoter
Petunia CHS-A promoter
Apple CHS1 promoter

dependent variables

Not explicitly mentioned

control variables

Arabidopsis ecotype Columbia
V26 genomic DNA
Malus domestica Royal Gala

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