IGEM:Harvard/2009/Notebook/Harvard iGEM 2010/2010/07/08

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Team Flavor Re-submitted sequencing order, with correct primers, to GeneWiz Did a confirmation digest of the Miraculin/Brazzein + YFP-2x constructs Ladder B15 E/X B15 S/P Mira+YFP N1 Mira+YFP N2 Mira+YFP C1 Mira+YFP C2 Brazz+YFP N1 Brazz+YFP N2 Brazz+YFP C1 Brazz+YFP C2 Ladder It appears that the digestion of Mira+YFP C2 (lane 7) did not work properly. This could have been the result of choosing a bacterial colony that was the result of contamination, or from simply an error in the digestion step. Transformed two pDUET expression vectors for Miniprep Ligated Miraculin/Brazzein to a StrepII tag Ligation Reactions Miraculin xbaI/pstI w/ B15 speI/pstI Miraculin ecoRI/speI w/ B15 ecoRI/xbaI Brazzein xbaI/pstI w/ B15 speI/pstI Brazzein ecoRI/speI w/ B15 ecoRI/xbaI Control B15 speI/pstI Control B15 ecoRI/xbaI DNA Insert 3 4 4 2 0 0 T4 DNA ligase buffer (10x) 2 2 2 2 2 2 diH2O 11 10 10 12 14 14 T4 DNA ligase 1 1 1 1 1 1 DNA Backbone 3 3 3 3 3 3 We then transformed the ligated plasmids into Turbo e. coli and plated the bacteria on Amp plates. Plates were left in the 37°C incubator overnight. Team Fence After receiving the new primers we performed PCR on barnase vectors PMT413 and PMT1002, LacIN, as well as the GAL4 DNA binding domain. The ladder is in lane 2, the GAL4 PCR products are in lanes 5 through 9, the barnase PMT413 lanes 10 through 14, and the LacIN PCR product is in the last 3 lanes. After PCR was confirmed successful, we decided to gel purify the LacIN because of the multiple band sizes seen in the gel. Below is the gel where LacIN was gel extracted, the chunk missing from the gel is where LacIN was identified to be. Gel extraction was performed according to the QIAgen gel extraction kit protocol using a microcentrifuge. Barstar Sequencing Submited plasmids from each of the 7 Barstar colonies for sequencing. Team Allergy amiRNA Sent out plasmids for sequencing w/ new primers Allergen Panel Sent out for sequencing this morning using new primers Minipreps of Allergen Panel Concentrations: LTP 1,2,3: 265.5 ng/uL, 507.1 ng/uL, 275.5 ng/uL; GerA 1,2,3,4: 283.1 ng/uL, 185.9 ng/uL, 182.4 ng/uL, 314.7 ng/uL; Bet 2A 1,2,3,4: 246.6 ng/uL, 251.4ng/uL, 550.3 ng/uL, 235.8 ng/uL; Bet 2S 1,2,3,4,5,6: 613 ng/uL, 296.8 ng/uL, 333.1 ng/uL, 467.3 ng/uL, 230.3 ng/uL, 129.4 ng/uL; Bet 1A 1,2,3,4,5: 505.8 ng/uL, 632.1 ng/uL, 561.7 ng/uL, 594.4 ng/uL, 609.2 ng/uL Diagnostic Digest PDK intron Gradient PCR of PDK intron Gel of gradient PCR of the 741bp PDK intron out of pHANNIBAL and pKANNIBAL. Lanes are (left to right, temperatures are annealing temperatures used in the gradient PCR) 1kb+ ladder, pHAN 50ºC, pKAN 50ºC, pHAN 55ºC, pKAN 55ºC, pHAN 60ºC, pKAN 60ºC, pHAN 65ºC, pKAN 65ºC. It appears that pHANNIBAL worked at 50, 55ºC but pKANNIBAL didn't. Concentration: hannibal pdk 12.7 ng/uL kannibal pdk: 15.6 ng/uL Did a PCR of the PCR product Annealing Temps: hannibal pdk (50 C); kannibal pdk (55 C) Extension Time: 30 sec 12 reactions (6 hannibal pdk; 6 kannibal pdk) Name Amount (uL) 10mM dNTPs 1 5x Phusion Buffer 10 Phusion Polymerase .5 pdk 1 uL DNA (12.6 and 16.5 ng) Fwd/Rev Primer .5 uM each (1uL 50x) at .5 uM Water 35.5 Growing up 5 mL cultures of pHannibal and pKannibal for getting more DNA for pdk pcr Gel purifying/digesting/ligating/transforming pdk intron into e.coli