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

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Ligation of Brazzein & Miraculin (w/ YFP tags) into DUET vector
Digestion


 * 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

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

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