IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/21

UIUC - Arsenic Collection System!

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7/21/10 Sold iGEM Dippin' Dots most of the day!!! 3:50 PM Miniprepped yesterday's cultures of gas vesicles and promoters J23100/J23119. Diluted to 20 ng/μL for PCR. 5:00 PM PCR Ingredient Amount 10X buffer 2 μL 10 mM dTNPs .4 μL Template 1 ul (20 ng) V2R Primer .4 μL VF Primer .4 μL Polymerase .2 μL H2O 15.6 μL Samples: J23100-Gas Vesicles-Kan Backbone J23119-Gas Vesicles-Kan Backbone Key1Tet (Steve) Program: 95°C 2 min 95°C 30 sec 55 °C 30 sec 72 °C 6 min Go to step 2 31X 72 °C 10 min hold at 4°C 6:20 PM Digestions pRpoS438 DNA 16.39 μL H2O 26.11 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL pArsRF1R1 DNA 17 μL H2O 25.5 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL pSB1A2 (2X) DNA 1.82 μL H2O 40.68 μL Buffer 2 5 μL BSA .5 μL XbaI 1 μL SpeI 1 μL Incubate at 37°C for 15 min, inactivate at 80°C for 20 min. 7:35 PM Gel for Band Extraction Lane Sample Amount 1 1 Kb Ladder 1 μL 2 pSB1A2 (1) 25 μL + 1 μL dye 3 pSB1A2 (1) 25 μL + 1 μL dye 4 pSB1A2 (2) 25 μL + 1 μL dye 5 pSB1A2 25 μL + 1 μL dye Made 5 mL cultures of plates from this morning (ArsR-Amp): the NEB plate had much more growth compared to the plate we used our competent cells on. 8:00 PM Plated out Gas Vesicles-Promoters again on new Kan plates (200 μL). 9:05 PM Band Extraction Concentrations (pSB1A2): 16.3 ng/μL and 12.6 ng/μL 9:15 PM SAP 1.2 μL SAP buffer .5 μL SAP ~10-12 μL pSB1A2 digest incubated 20 min at 37°C, inactivated 15 min at 65°C 9:35 PM Gel Lane Sample Amount 1 1 kB 1 μL 2 J23100-gv-kan 5 μL + 1 μL dye 3 J23119-gv-kan 5 μL + 1 μL dye 4 Key1tet 5 μL + 1 μL dye 10:10 PM pRpoS438-pSB1A2 H2O 13 μL pRpoS 2 μL pSB1A2 2 μL Ligase Buffer 2 μL Ligase 2 μL pArsR-pSB1A2 H2O 13 μL pArsR 2 μL pSB1A2 2 μL Ligase Buffer 2 μL Ligase 2 μL Room temp incubation 10 min, inactivation 80°C 20 min. 10:50 PM Transformation 50 μL NEB electrocompetent 5α cells, 3 μL ligation product. electroporate, 1 mL LB, recover at 37°C for 1 hour. 11:50 PM plated 200 ul cells on Amp plates.