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):

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

Gel


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

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:



Sequencing

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