The MPK1 genomic region was amplified from Col-0 genomic DNA (Thole et al., 2014 (link)) using Pfx Platinum Taq (Life Technologies) with caccMPK1-URR and MPK1g-nostop2. The resultant 2897-bp PCR product contained the MPK1 upstream region and entire MPK1 coding region (from –1693 to 1198, where the A of the ATG start codon is position 1) with the TGA stop codon replaced with a GGA codon encoding for Gly. This PCR product was captured into the pENTR/D-TOPO vector (Life Technologies). The pENTR-DTOPO-MPK1:MPK1nostop vector was linearized by digestion with MluI. The MPK1:MPK1nostop genomic region was recombined into the pMDC107 plasmid (Curtis and Grossniklaus, 2003 (link)) using LR Clonase (Life Technologies) to form MPK1:MPK1-GFP, which expresses a C-terminal GFP fusion with MPK1 driven by the region upstream of MPK1.
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Cloning C-terminal MPK1-GFP Fusion
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Corresponding Organization : Washington University in St. Louis
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Variable analysis
independent variables
- Genomic region (MPK1) amplified from Col-0 genomic DNA
- Expression of MPK1-GFP fusion protein
- Pfx Platinum Taq (Life Technologies) used for PCR amplification
- PENTR/D-TOPO vector (Life Technologies) used for capturing PCR product
- PMDC107 plasmid (Curtis and Grossniklaus, 2003) used for recombination
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