IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/03

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Result from bacillus transformation with fresh competent cells (02/09)

strain antibiotic added vector observation conclusion
IA751 none none confluent colonies cells are alive
IA751 Spc50 none no colonies no contamination
IA751 Spc50 ECE153 70 colonies transformation to check, efficiency : about 115 colonies/μg of DNA
IA771 none none confluent colonies cells are alive
IA771 Cm5 none no colonies no contamination
IA771 Cm5 ECE166 16 colonies transformation to check, efficiency : about 27 colonies/μg of DNA
IA771 Cm5 ECE112 41 small colonies transformation to check, efficiency : about 41 colonies/μg of DNA

With 1μg of DNA, we manage to obtain colonies with ECE112. We will check our transformation with starch plates.

Check ECE112 stock

- Single and double digest with XbaI and EcoRI

Gel3.gif

- Gel

  • Lane 3 : HyperladderI
  • Lane 4 : ECE112, 100mL flask, single digest with XbaI (expected size about 10,000bp)
  • Lane 5 : ECE112, 100mL flask, single digest with EcoRI (expected size about 10,000bp)
  • Lane 6 : ECE112, 100mL flask, double digest (expected size about 3200bp and 6,900bp)
  • Lane 7 : ECE112, 10mL flask, single digest with XbaI (expected size about 10,000bp)
  • Lane 8 : ECE112, 10mL flask, single digest with EcoRI (expected size about 10,000bp)
  • Lane 9 : ECE112, 10mL flask, double digest (expected size about 3200bp and 6,900bp)
  • Lane 10 : HyperladderI

- Results : ok

Test glycerol stocks (from 02/09) of competent bacillus

strain antibiotic added vector quantity of DNA
IA751 none none 0
IA751 Spc50 none 0
IA751 Spc50 ECE153 0.6
IA771 none none 0
IA771 Cm5 none 0
IA771 Cm5 ECE166 0.6

Starch plates

- Use blank plates

- Melt 100mL of Soft Agar, and add slowly 1g of starch

- Mix, boil to sterilize

- Pour on blank plate

- Put 4 or 5 colonies on each plate

  • IA751 from blank plate (positive control)
  • IA771 from blank plate (negative control)
  • IA751 transformed with ECE112 (Cm5, 29/08)
  • IA751 control (Cm5, 29/08)
  • IA751 + ECE112 (Cm5, 02/09)
  • IA751 + ECE153 (Cm5, 02/09)
  • IA751 + ECE153 (Cm5, 29/08)