IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/12

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Team Allergy

Today, we received sequences for the allergen , intron, and amiRNA inserts. Since 4 of the 8 allergen inserts were not correctly done (and were not set for sequencing) and we want to repeat the inserts that DID work, we will redo ligations for the entire panel.

Procedures

  1. Check Sequencing Results
  2. Redo Ligations for allergen Panel

Results

Sequencing Results

  • amiRNA
    • GFP #1 & GFP.5 #3 worked
    • Bet,LTP did not work
  • intron
    • Pal(#2) matched
  • ihpRNA
    • LTP Sense 1,2,3 and Ger Sense 1 matched
    • Bet sense did not match

Full report (annotated sequences): File:Report.zip

Redo Ligations w/ phosphatized backbone for entire allergen panel,

PME did not grow, we will not use it since we have good PDK and PAL introns.

PAL and PDK will be digested and inserted into a plasmid containing a sense strand of an allergen.

Add PDK to amiRNA vectors if sequences match up

Team Flavor

  • We removed our cultures of e. coli containing Miraculin/Brazzein C/N YFP and NosT & Stop and e. coli containing Miraculin/Brazzein and StrepII tag from the 37°C shaker.
  • Glycerol stocks were made (666 μL glycerol and 333 μL cells)

Miniprep

   ODs
    Miraculin C YFP and NosT & Stop 1: 414.4 ng/μL
    Miraculin C YFP and NosT & Stop 2: 304.0 ng/μL
    Miraculin N YFP and NosT & Stop 1: 428.6 ng/μL
    Miraculin N YFP and NosT & Stop 2: 421.0 ng/μL
    Brazzein C YFP and NosT & Stop 1: 572.8 ng/μL
    Brazzein C YFP and NosT & Stop 2: 580.3 ng/μL
    Brazzein N YFP and NosT & Stop 1: 348.9 ng/μL
    Brazzein N YFP and NosT & Stop 2: 639.6 ng/μL
    Miraculin N StrepII 1: 888.6 ng/μL
    Miraculin N StrepII 2: 614.4 ng/μL
    Miraculin C StrepII 1: 677.1 ng/μL
    Miraculin C StrepII 2: 222.1 ng/μL
    Brazzein N StrepII 1: 352.6 ng/μL
    Brazzein N StrepII 2: 331.6 ng/μL
    Brazzein C StrepII 1: 209.3 ng/μL
    Brazzein C StrepII 2: 346.4 ng/μL


Digestion

Digestion Reactions
Miraculin C YFP NST Miraculin N YFP NST Brazzein N YFP NST Brazzein C YFP NST
diH2O 14 14 13.5 14.3
Green FD buffer (10x) 2 2 2 2
DNA 2 2 2.5 1.7
NotI 1 1 1 1
SpeI 1 1 1 1


Gel

Mira brazz+YFP 7 12 2010.jpg

   Gel Lanes:
    1. 1 kb plus ladder
    3. Miraculin N YFP NST (NotI/SpeI)
    5. Miraculin C YFP NST (NotI/SpeI)
    7. Brazzein N YFP NST (NotI/SpeI)
    9. Brazzein C YFP NST (NotI/SpeI)
    11. 1 kb plus ladder


  • Gel purify per Qiagen protocol
   ODs
    Brazzein C YFP NST (NotI/SpeI): 6.3 ng/μL
    Brazzein N YFP NST (NotI/SpeI): 8.5 ng/μL
    Miraculin C YFP NST (NotI/SpeI): 3.6 ng/μL
    Miraculin N YFP NST (NotI/SpeI): 12.8 ng/μL


Team Fence

Nanodrop of Barnase, Gal4 and LacIN Minipreps

Plasmid Quantity (ng/μL) 260/280
Barnase 1 226.1
Barnase 2 336.3 1.94
Gal4 1 298.0 1.88
Gal4 2 363.3 1.92
Gal4 3 301.9 1.89
LacIN 1 376.7 1.89
LacIN 2 237.8 1.90
LacIN 3 339.4 1.92
LacIN 4 368.9 1.92


Digestion of Ligation product minipreps

Xba1 and Pst1

Mastermix (10x)

  • 10μL Xba1
  • 10μL Pst1
  • 20μL Buffer


Barnase 1

  4μL Barnase 1
  12μL DH2O
  4μL Mastermix

Barnase 2

  3μL Barnase 2
  13μL DH2O
  4μL Mastermix

Gal 1

  3μL Gal 1
  13μL DH2O
  4μL Mastermix

Gal 2

  2.5μL Gal 2
  13.5μL DH2O
  4μL Mastermix

Gal 3

  3μL Gal 3
  13μL DH2O
  4μL Mastermix

LacIN 1

  2.5μL LacIN 1
  13.5μL DH2O
  4μL Mastermix

LacIN 2

  4μL LacIN 2
  12μL DH2O
  4μL Mastermix

LacIN 3

  2.5μL LacIN 1
  13.5μL DH2O
  4μL Mastermix

LacIN 4

  2.5μL LacIN 1
  13.5μL DH2O
  4μL Mastermix


Put tubes in 37°C heat bath for 30 mins, then put them on ice.

Gel of Digestions:

07122010ve3ctordigestmirbraz2.jpg


Sequencing

  • submitted Barnase 1 and 2, Gal4 1,2, and 3, and LacIN 1,2,3, and 4 for sequencing