IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/01

UIUC - Arsenic Collection System!

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7/1/10 Meagan plated the cells transformed last night at 12:30 AM. We observed no growth on the plates this morning when we checked at 9:30 AM for both those plates and the PAL_pSB1A2 plate. 11:15 AM Troubleshooting Gel Ran ligations and digests on gels at 80V. Lane Sample Amount 1. 1 Kb Ladder 2 ul 2. J23100 digest 5 ul sample, 1 ul dye 3. J23119 digest 5 ul sample, 1 ul dye 4. Gas Vesicles digest 5 ul sample, 1 ul dye 5. Tet Backbone digest 5 ul sample, 1 ul dye 6. J23100-Gas vesicle ligation 5 ul sample, 1 ul dye 7. J23119-Gas vesicle ligation 5 ul sample, 1 ul dye 8. pSBIT3 5 ul sample, 1 ul dye 9. PAL digest 5 ul sample, 1 ul dye 10. PAL ligation 5 ul sample, 1 ul dye Lane Sample Amount 1. 1 Kb Ladder 2 ul 2. pSB1A2 EcoRI/PstI 5 ul sample, 1 ul dye 3. pSB1T3 EcoRI/PstI 5 ul sample, 1 ul dye 4. pSB1C3 EcoRI/PstI 5 ul sample, 1 ul dye 5. pSB1K3 EcoRI/PstI 5 ul sample, 1 ul dye 6. pSB1A2 XbaI/SpeI/SAP 5 ul sample, 1 ul dye 7. pSB1T3 XbaI/SpeI/SAP 5 ul sample, 1 ul dye 8. pSB1C3 XbaI/SpeI/SAP 5 ul sample, 1 ul dye 9. pSB1K3 XbaI/SpeI/SAP 5 ul sample, 1 ul dye 10. pSB1K3 5 ul sample, 1 ul dye 3:50 PM Re-Ligating J23100-Gas Vesicles-pSB1T3 H20 11 ul J23100 2 ul Gas Vesicles 2 ul pSB1T3 2 ul T4 buffer 2 ul T4 Ligase 1 ul J23119-gas vesicles-pSB1T3 H2O 11 ul J23119 2 ul gas vesicles 2 ul pSB1T3 2 ul T4 Buffer 2 ul T4 Ligase 1 ul Gas Vesicles-pSB1T3 H20 12 l Gas Vesicles 2 ul pSB1T3 2 ul T4 buffer 2 ul T4 Ligase 1 ul incubate at room temperature for 10 minutes inactivate at 80°C for 20 minutes 5:15 PM Transformations 100 ul cells + 5 ul ligation product (J23100-Gas Vesicles-pSB1T3 or J23119-Gas Vesicles-pSB1T3). electroporate and add 1 mL LB, incubate at 37°C for 1 hour for recovery. 6:30 PM Plating Plated 20ul transformation product on 1/2 tetracycline plate, plated 200 ul on other half. Next day observed no growth.