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 Check Sequencing Results 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 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: Sequencing submitted Barnase 1 and 2, Gal4 1,2, and 3, and LacIN 1,2,3, and 4 for sequencing