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

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Results from yesterday

 * Test of antibiotic resistances of strains of last year


 * Transformation of E.coli with different plasmids from last year

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 ]

Dilution of Cm. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]


 * 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 -Aliquot into 5 different bottles. [180mL each]
 * Yeast Extract 10g
 * Casamino Acid 2g
 * add Distilled water to 900mL

10x Bacillus Salts -Aliguot into 5 different bottles. [200mL each]
 * NH4)2SO4 20g
 * K2PO4 anhydrous 139.66g
 * KH2PO4 60g
 * Tri-Sodium Citrate 10g
 * MgSO4.7H2O 2g
 * Add Distilled water to 1000mL

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]

-Total volume 200mL
 * Add 100mL of 50mM CaCl2.2H2O
 * Add 100mL of 250nM MgCl2.6H2O

NOTE: To complete Medium B, Take 0.2mL of this solution


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