IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/05
 |
 Main project page Previous entry Next entry
| |||||||||
Checking vectors- Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153 - Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday) - Run on a gel single digest for ECE147 (from yesterday with more DNA), ECE149 (from yesterday with more DNA), ECE150 (from yesterday with more DNA) and ECE 153, ECE162, ECE172
 Very low bands : not enough DNA!!! Transformation of Bacillus
- Prepare medium A with tryptophan, and medium B - add 5mL of medium A in 3 different tubes, inoculate each tube with colonies (1A1, IA751 ans IA771) - Check OD every 20min - Incubate 90min - Add 0.45mL of medium B and 0.05mL of culture in Ependorf tubes - Incubate 90min - Transform : add 1μg of DNA (some with ECE112, some with ECE166) (so you need to nanodrop samples before!) - Incubate 30min - Pipette 200μL of solution, spread it on aech plate, wait 10min, and do it again - Incubate 24hours - A few glycerol tubes to stock cells : add 2/7 glycerol to cell tubes
- Spin glycerol stocks, pipette out glycerol - Add 0.5mL of medium B, incubate for 1 hour - Add 10μL of ECE112 (640ng) - Incubate 2hours - Plate 200μL, and 10min later, still 200μL
New stocks- Do glycerol stock of I746001 and I746101 (no sterile glycerol) - Put IA751, IA771 in 10mL LB - Put ECE 176 in 10mL LB + antibiotic - Reinoculate the tube of LB from yesterday with ECE166 plate - ECE176 replated onto Amp100 + Cm5
| ||||||||||
