IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/03: Difference between revisions

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We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.
We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.
Picture of the gel with the different promoters
[[Image:gel5.gif|300px|center]]


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Revision as of 07:43, 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

Checking the insert of promoters

We ligated promoters into death vector, and transforned into Top 10. Our transformation worked, but it was hard to say with the result of yesterday if we had the good insert. Moreover, it is possible that we inverted Pupp and Pspac. So we are going to make a new single colony PCR with the primers of promoters (we used the same single colonies than yesterday).

We also want to PCR again all promoters (Pspac, Pxyl, Pupp and Ppac) from plasmids to have more.

Picture of the gel with the different promoters


promoter colony primers name
Pspac 1 Pspac primers A
Pspac 1 Pupp primers B
Pspac 2 Pspac primers C
Pspac 2 Pupp primers D
Pupp 1 Pupp primers E
Pupp 1 Pspac primers F
Pupp 3 Pupp primers G
Pupp 3 Pspac primers H

- Single colony PCR : add 10μL of SDW, 5μL of MM, 1μL + 1μL of primers, 3μL of cells

- Gel

  • lane 3 : hyperladderI
  • lane 4 : Pspac (colony1) with Pspac primers
  • lane 5 : Pspac (colony1) with Pupp primers
  • lane 6 : Pspac (colony2) with Pspac primers
  • lane 7 : Pspac (colony2) with Pupp primers
  • lane 8 : Pupp (colony1) with Pupp primers
  • lane 9 : Pupp (colony1) with Pspac primers
  • lane 10 : Pupp (colony3) with Pupp primers
  • lane 11 : Pupp (colony3) with Pspac primers
  • lane 12 : HyperladderI

- Results

promoter colony primers observation Conclusion
Pspac 1 Pspac primers nothing it is in fact Pupp
Pspac 1 Pupp primers band (about 280bp) Pupp OK
Pspac 2 Pspac primers nothing it is in fact Pupp
Pspac 2 Pupp primers band (about 280bp) Pupp OK
Pupp 1 Pupp primers nothing it is in fact Pspac
Pupp 1 Pspac primers band (about 180bp) Pspac OK
Pupp 3 Pupp primers nothing it is in fact Pspac
Pupp 3 Pspac primers band (about 180bp) Pspac OK

Make some stock of our new biobricks

  • Pupp (colony1) into the death vector, transformed into TOP10
  • Pspac (colony1) into the death vector, transformed into TOP10
  • agrD (colony5) into the death vector, transformed into TOP10

- Grow this single colony into 10mL of LB (Cm35)

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)

Transformation of new "biobricks"

- transform into E.coli :

  • agrA
  • agrB
  • agrC
  • Pxyl


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