Generation of pDRf1-GW
Lab/Group: Wolf Frommer lab (Carnegie Institution, Stanford)
Related Journal & Article Information
Journal: Nature
Introduction
For rapid cloning of genes of interest into a yeast expression vector, the GatewayTM technology can be used.
Materials
Reagents
Equipment
Procedure
1) pDRf1-GW is a yeast shuttle vector carrying a GATEWAYTM cloning cassette (Invitrogen, Carlsbad, CA) and an f1 viral replication origin. Generate pDRf1-GW by substituting a SpeI-SalI fragment from pDR1961 with a new multi-cloning sequence (SpeI, EcoRI, NotI, PstI, SalI).
2) Amplify by PCR a 500bp DNA fragment containing the f1 replication origin from pGEM-T-easy (Promega, Madison, WI). Use primers containing a HindIII restriction site, and then clone into the HindIII site located upstream of the PMA1 promoter fragment to generate the pDRf1 vector.
3) Amplify the GATEWAYTM fragment by PCR with primers containing an EcoRV site and clone in a blunt-ended NotI site in pDRf1 to generate pDRf1-GW.
Troubleshooting
Critical Steps
Anticipated Results
References
Rentsch, D. et al. NTR1 encodes a high affinity oligopeptide transporter in Arabidopsis. FEBS Lett. 370, 264-268 (1995).
Acknowledgements
Keywords
Yeast vector, Gateway compatible