IGEM:Cambridge/2008/Notebook/Turing Pattern Formation/2008/08/20

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Check Lux components

- Single colony PCR for :

  • R0040 (MIT stuff)
  • R0062 (MIT stuff)
  • 4 different colonies of S0168 (from a transformation plate from 12/08)
  • 4 different colonies of J04630 (from a transformation plate from 12/08)

- Protocol : add 1μL of cells (diluted in water), 10μL of Master Mis, 7μL of SDW, 1μL of VF primer and 1μL of VR primer

- Gel PCR products

Gel 1

  • Lane2 : Hyperladder1
  • Lane3 : JO4630, colony 1
  • Lane4 : JO4630, colony 2
  • Lane5 : JO4630, colony 3
  • Lane6 : JO4630, colony 4
  • Lane7 : HyperladderI

Gel 2

  • Lane2 : HyperladderI
  • Lane3 : ECE190 double digest
  • Lane4 : S0168, colony 1
  • Lane5 : S0168, colony 2
  • Lane6 : S0168, colony 3
  • Lane7 : S0168, colony 4
  • Lane8 : HyperladderI
  • Lane9 : R0040
  • Lane10 : R0062
  • Lane11 : Ladder 100bp

- Results

  • R0040 and R0062 : one big band of about 300b (expected size 293), OK!
  • S0168 : one band of about 400b for the 4 different colonies (expected size 1234!), bad! This plate does not contain S0168
  • J04630 (colonies 2 and 4) : one band of about 1100b (expected size 1173), OK!
  • J04630 (colony 1) : one good band plus another band...
  • J04630 (colony 3) : one band of about 600b, bad!


Ligation

- Materials :

  • AgrA
  • AgrB
  • AgrC
  • AgrD
  • Pupp
  • Pspac
  • Ppac
  • Pxyl
  • RBS S
  • RBS W
  • psB4C5

- Double digest of PCR products

- Run vector, AgrA and AgrD on a gel

- DNA clean and concentrator for AgrA, B,C and D, promoters

- Microclean for both RBS

- Nanodrop

260/280 ng/μL
AgrA 1.66 16.4
AgrB 1.91 23.5
AgrC 1.99 35.9
AgrD 2.13 4.9
Pxyl 1.54 5.6
Ppac 1.49 4.6
Pspc 1.62 9.6
Pupp 1.88 8.5
RBS S 2.44 29
RBS W 1.44 10.7

- Extract plasmid annd Agr from gel and clean

- Ligation


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