IGEM:Cambridge/2008/Notebook/Bacillus/2008/07/24

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Results from yesterday Test of antibiotic resistances of strains of last year Strain Plasmid Extracted from Antibiotic use Observations Conclusion DH5alpha PP182 tube Amp Many colonies Amp Resistance OK DH5alpha PP182 plate Amp Many colonies Idem MC1061 NONE tube Cm Nothing As expecting, no Cm Resist. MC1061 NONE plate Cm Nothing Idem MC1061 PNZ8901 plate Cm Maybe a few colonies Contamination?? MC1061 PNZ8901 tube Cm No colonies no Cm Resist. Transformation of E.coli with different plasmids from last year Strain Inserted plasmid Antibiotic Observation Conclusion E.coli I746000 Amp No colonies Antibiotic resistance unknown, no Amp Resist., Pb : no terminator E.coli I746100 Amp No colonies Antibiotic resistance unknown, no Amp Resist., Pb : no terminator E.coli I746001 Amp Many colonies Transformation OK E.coli I746101 Amp Many colonies Transformation OK Antibiotic test for Bacillus resistance We want to find the lowest concentration of antibiotic which kills Bacillus. Dilution of Amp. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ] Concentration (μg/mL) 100 75 50 25 10 100 mg/mL Stock 1:1000 3:4000 1:2000 1:4000 1:10000 Dilution of Cm. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ] Concentration (μg/mL) 35 25 15 10 5 100 mg/mL Stock 1:1000 1:1400 3:7000 1:3500 1:7000 Add Disks in antibiotic solution for 5 mins [ Protocol to sterilize tweezers : Wipe with Kimwipes, Ethanol, Flame ] Melt Soft Agar 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08 Pour SA+Cells over blank hard agar plates put disks with different concentration of Amp/Cm above SA Incubate at 37°C Results [Plates were prepared before lunch, at 5pm there were visible growth] -Amp Plates: Huge rings of no growth around 100, 75, 50, 25, and 10. -Cm PLates: Tiny rings of no growth around 5, 10, 15 and 25. Small ring of no growth around 35 but not well defined. (No clear zones) Salts for making Bacillus Competent 10x Medium A Base Yeast Extract 10g Casamino Acid 2g add Distilled water to 900mL -Aliquot into 5 different bottles. [180mL each] 10x Bacillus Salts NH4)2SO4 20g K2PO4 anhydrous 139.66g KH2PO4 60g Tri-Sodium Citrate 10g MgSO4.7H2O 2g Add Distilled water to 1000mL -Aliguot into 5 different bottles. [200mL each] Medium B Preparing 50mM CaCl2.2H2O CaCl2.2H2O 1.470g Add Distilled water to 20mL [Final conc. 500mM] Take 10mL of 500mM Add 90mL of distilled water [Final conc. 50mM] Preparing 250nM MgCl2.6H2O MgCl2.6H2O 0.508g Add Distilled water to 10mL [Final conc. 250mM] Take 1mL of 250mM Add 99mL of Distilled water [Final conc. 250μM] Take 1mL of 250μM Add 99mL of Distilled water [Final conc. 250nM] Add 100mL of 50mM CaCl2.2H2O Add 100mL of 250nM MgCl2.6H2O -Total volume 200mL NOTE: To complete Medium B, Take 0.2mL of this solution