Generation of AMT1;1-GFP
Lab/Group: Wolf Frommer lab (Carnegie Institution, Stanford)
Related Journal & Article Information
Journal: Nature
Introduction
To study the subcellular localization of the ammonium transporter, GFP fusions can be generated.
Materials
Reagents
Equipment
Procedure
1) Introduce HindIII and XhoI sites by site directed mutagenesis 5’ and 3’ respectively of the eGFP ORF from pCMV-EGFP (InVitrogen, Carlsbad, CA).
2) Amplify the GATEWAYTM fragment by PCR with primers containing an EcoRV site in the forward primer and HindIII sites in the reverse primer and clone blunt in the SmaI restriction site of pDR196.
3) Digest this plasmid by HindIII to isolate a pPMA-GW fragment and ligate with a HindIII-eGFP-XhoI fragment into the HindIII-XhoI sites of pDR196 to generate a pDR-GW-eGFP plasmid. 4) Clone AtAMT1;1 into pDR-GW-eGFP using GATEWAYTM technology to generate pDR-AtAMT1;1-eGFP.
5) Confirm the AtAMT1;1-eGFP C-terminus fusion by sequencing and test for functionality by the yeast complementation assay in the yeast strain 31019b (see yeast growth assays protocol). The AtAMT1;1-eGFP complements the mutants deficient in ammonium uptake (data not shown).
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
Yeast, eGFP