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.

- Single and double digest with XbaI and EcoRI

- 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

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

- 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