IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/09

iGEM iGarden

Main project page Previous entry      Next entry

Team Flavor Ligation of Brazzein & Miraculin (w/ YFP tags) into DUET vector Digestion Digestion Reactions Brazzein & YFP C EcoRI/SpeI Brazzein & YFP N EcoRI/SpeI Miraculin & YFP C EcoRI/SpeI Miraculin & YFP N EcoRI/SpeI V24 NotI/SpeI NosT & Stop EcoRI/XbaI DNA 2 2 2 3 5 2 FD Buffer (10x) 2 2 2 2 2 2 diH2O 14 14 14 13 11 14 Enzyme 1 1 1 1 1 1 Enzyme 1 1 1 1 1 1 We are adding the NosT & stop to the C-terminus of the Miraculin and Brazzein constructs. This whole construct will then be ligated into the V24 DUET vector. Gel Gel Lanes: 1. 1kb plus ladder 3. Miraculin & YFP N-terminus ecoRI/SpeI 5. Miraculin & YFP C-terminus ecoRI/SpeI 7. Brazzein & YFP N-terminus ecoRI/SpeI 9. Brazzein & YFP C-terminus ecoRI/SpeI 11. V24 NotI/SpeI 13. NosT & Stop EcoRI/XbaI Team Fence Digest of yesterday's ligations LacIN - 65μL 1μL Xba1 1μL Pst1 6.5μL FD buffer 1μL DPN1 50μL LacIN 6.5μL DH2O Gal4DBD - 50μL 1μL Xba1 1μL Pst1 7μL Gal4 1μL DPN1 5μL FD Buffer 36μL DH2O Barnase - 50μL 1μL Xba1 1μL Pst1 15μL Barnase 1μL DPN1 5μL FD buffer 28μL DH2O Put in 37°C bath for 30 mins Following proceedure for PCR cleanup to clean up the digestion results added 5 volumes PB buffer to 1 volume digestion product 325μL for LacIN, 250μL for Gal4DBD and Barnase pippetted each into a QIAquick column, spun for 30 secs discarded flow through, put columns back in same tube, added 750μL PE buffer spun for 30 secs, discared flow through Spun again for 1 min placed column in new eppendorf added 30μL EB buffer to elute, spun for 1 min Eppendorfs labeled "LacIN digest cleanup 7/9," "Gal4 digest cleanup 7/9," and "Barnase digest cleanup 7/9" Nanodrops of LacIN, Gal4 and Barnase digestion cleanups