IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/09/02: Difference between revisions

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- Double digest with XbaI and EcoRI
- Double digest with XbaI and EcoRI
[[Image:gel2.gif|200px|center]]


- Gel : problem only one band (single digest???)
- Gel : problem only one band (single digest???)

Revision as of 07:32, 5 September 2008

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Transformation of Bacillus

  • with fresh plates from yesterday, last protocol
Time (min) 0 20 40 60 85 105 125
OD650 for IA751 0.1192 0.1330 0.1736 0.2238 0.3124 0.4523 0.5113
OD650 for IA771 0.1144 0.1216 0.1593 0.2296 0.3476 0.4302 0.5565
  • t0 (IA751) = 100min
  • t0 (IA771) = 110min
  • Transformation

- IA751 on Cm5 and Spc 50 (control DNA less plates)

- IA751 + ECE112 (1μg of DNA) on Cm5

- IA751 + ECE153 (0.6μg of DNA) on Scp50

- IA771 on Cm5 (control)

- IA771 + ECE166 on Cm5

  • Glycerol stocks (about 20 tubes of each)


Check transformation in E.coli of our ligated products

- Single colony PCR with VF and VR : add 10μL of SDW, 5μL of MM, 1μL of VF, 1μL of VR and 3μL of cells

inserted products number of colonies to check (chemical transf.) number of colonies to check (electrop. transf.)
RBS S 3 2
RBS W 3 2
Pspac 2 2
Pupp 2 2
agrD 3 2

- Gel (3% of agarose)

  • Result
gel lane inserted product (name of the colony) observation conclusion
top 3 RBS S (1) one band (260bp) size of the insert, RBS too small to see on a gel
top 4 RBS S (2) one band (260bp) size of the insert, RBS too small to see on a gel
top 5 RBS S (4) nothing ?
top 6 RBS S (5) nothing ?
top 7 RBS W (1) 2 band (260bp + 350bp) size of the insert, RBS too small to see on a gel + something else?
top 8 RBS W (2) one band (350bp) too big
top 9 RBS W (4) one band (260bp) size of the insert, RBS too small to see on a gel
top 10 RBS W (5) one band (260bp) size of the insert, RBS too small to see on a gel
top 11 Pspac (1) 2 band (260bp + 350bp) maybe problem with products loaded on gel (exactly the same bands than for RBS W)
top 12 Pspac (2) one band (450bp) size of Pupp?
top 13 Pspac (3) one band (450bp) size of Pupp?
bottom 3 Pspac (4) nothing ?
bottom 4 Pupp (1) one band (400bp) size of Pspac?
bottom 5 Pupp (2) one band (400bp) size of Pspac?
bottom 6 Pupp (3) one band (slightly lower than 400bp) size of Pspac?
bottom 7 Pupp (4) one band (400bp) size of Pspac?
bottom 8 agrD (1) 2 bands (380 and 450bp) big band is ok
bottom 9 agrD (2) 2 bands (380 and 450bp) big band is ok
bottom 10 agrD (3) no bands ?
bottom 11 agrD (4) strong band (450bp) ok
bottom 12 P2 one band (350bp) ok

Check ECE112 stock

- Double digest with XbaI and EcoRI

- Gel : problem only one band (single digest???)


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