IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/03: Difference between revisions

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(Autocreate 2008/09/03 Entry for IGEM:Cambridge/2008/Notebook/Bacillus)
 
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==Entry title==
==Check ECE112 stock==
* Insert your content here.


- Single and double digest with XbaI and EcoRI


[[Image:gel3.gif|300px|center]]


<!-- ## Do not edit below this line unless you know what you are doing. ## -->
- 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==
 
{|class="wikitable" style="text-align:center" border="1"
|-
! 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
|}
 
==Result from bacillus transformation with fresh competent cells (02/09)==
 
{|class="wikitable" style="text-align:center" border="1"
|-
! 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.
==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


__NOTOC__
* 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)

Revision as of 09:15, 5 September 2008

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Check ECE112 stock

- 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

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

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.

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)
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