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

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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 22
diH2O 14 14 14 13 11 14
Enzyme 1 1 1 1 1 1
Enzyme 1 1 1 1 11
  • 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


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