IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/06/29

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Team Allergy Today, we did not do work with amiRNA and focused our efforts on hpRNA. We obtained undigested V0120 with a death gene insert from Team Flavor, and we have inserts from previous PCR reactions that all need to be digested and ligated into V0120. Procedures: Digestion of Allergen Panel and V0120 with Xba and Pst Ligation of Allergen inserts into Vector Backbone Transformation of E. Coli Results Digestions Digestion Reactions Bet GFP LTP LTP(.5) GFP(.5) Backbone (V0120 w/o a death gene) DNA 28 28 28 28 28 5 FD Buffer (10x) 4 4 4 4 4 2 diH2O 6 6 6 6 6 11 EcoRI 1 1 1 1 1 1 SpeI 1 1 1 1 1 1 Concentrations: Bet: 58.2 ng/uL; LTP (.5): 38.6 ng/uL; GFP (.5): 43.9 ng/uL; GFP: 29.1 ng/uL; LTP: 60.4 ng/uL; Backbone: 20.2 ng/uL Ligations for full amiRNA and V0120 (w/o death gene) note: .5 denotes multistep pcr reaction from yesterday in which .5 uL of part 3 of gfp/ltp were use in one of the giant assemblies of parts 1,2,3 w/ primers A&B Ligation Reactions Bet GFP LTP LTP(.5) GFP(.5) DNA Insert 1 1 1 1 1 T4 DNA ligase buffer 10 10 10 10 10 diH2O 7 7 7 7 7 T4 DNA ligase 1 1 1 1 1 DNA Backbone 1 1 1 1 1 Transformation ( see transformation protocol) Transformed amiRNA into turbo cells and plated on LB amp plates--will check for colonies tommorow (Plated at ~ 4pm) Team Fence B21 plasmid maxiprep colony inoculated overnight and medium was centrifuged to obtain a pellet. followed instructions as described in the QIAgen Plasmid Purification Handbook pg. 19-23. (2005 edition) Team Flavor Results of ligation and transformation of The Big Three (pENTCUP2, NosT, NosT + stop) with B15 backbone into E.coli A few colonies were found on the pENTCUP2 + Backbone plate, NosT + stop and backbone plate, and b15 backbone control plate. Colonies were picked from the plates and placed in 3ml of LB and agarose in a culture tube. The tubes were placed in the 37°C shaker for 8 hours. 2 mL of LB+AMP was added to the cultures, bring the total volume to 5 mL of LB+AMP. Cultures were left to shake overnight. DLT of the Sweethearts - Miraculin and Brazzein (and B21) Digestion Reactions Miraculin Brazzein B21 DNA 6 11 7 FD Buffer (10x) 2 2 2 diH2O 10 5 9 EcoRI 1 1 1 SpeI 1 1 1 Placed in 37°C waterbath for 20 minutes. Digestion Gel Gel Lanes: 1. 1 kb plus ladder 2. Miraculin digested with EcoRI/SpeI 3. Brazzein digested with EcoRI/SpeI 4. B21 digested with EcoRI/SpeI 5. 1 kb plus ladder Nanodrop Specs (from gel purification) Name Concentration (ng/μL) Miraculin 9.5 Brazzein 10.2 B21 Vector (V0120) 8.4 Ligation Reactions Miraculin Brazzein Control Insert 3.5 0.75 0 Vector 6 6 6 Buffer (10x) 2 2 2 Ligase 1 1 1 H2O 6.5 10.25 0 The ligation of Miraculin was done at a 3:1 ratio (insert to vector) due to its larger size (~700 bp) The ligation of Brazzein was done at a 2:1 ratio due to its smaller size (~200 bp)