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

- Extract plasmid annd Agr from gel and clean

- Ligation


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