IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/05

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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

  • Results
  • Lane3 : HyperladderI
  • Lane4 : ECE147
  • Lane5 : ECE149
  • Lane6 : ECE150
  • Lane7 : ECE151
  • Lane8 : ECE1162
  • Lane9 : ECE172
  • Lane10 : hyperladderI


Very low bands : not enough DNA!!!

Transformation of Bacillus

  • Result of Nanodrop
Vector 260/280 ng/μL
ECE112 1.75 64.6
ECE166 172 138.6

- 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


- Transformation from glycerol stock from 30/07/2008

- 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



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