Team:Evry/Interlab Study/08-26-2014

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Construction n°1: PSB1C3 with I20260


Loading of the PSB1C3 digestion by EcoRI and PstI from the 25th August, on a 1% agarose gel. The 40 µl were loaded with 8 µl of loading dye 6X. Migration was performed 1 hour at 110 mV.
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Figure 6: 1% agarose gel of colony PCR products for BBa_E0240 and BBa_I20260. Lane 2 and 3: digestion product of PSB1C3 by EcoRI and PstI, Lane 4: Purple 2-Log ladder NEB



Construction n°2: PSB1C3 with J23101-E1010




Construction n°3: pSB1C3 with K823012-E1010


Digestion of the insert pSB1C3 (J23115) with EcoRI and XbaI:
  • Steril H2O: 10,8µL
  • Buffer 2.1 NEB : 2µL
  • BSA: 0,2µL
  • DNA: 5µL
  • EcoRI-HF: 1µL
  • XbaI: 1µl

Digestion of the vector pSB1C3(E0240) with EcoRI and SpeI:
  • Steril H2O: 10,8µL
  • Buffer 2.1 NEB : 2µL
  • BSA: 0,2µL
  • DNA: 5µL
  • EcoRI-HF: 1µL
  • SpeI: 1µl

Mix were incubated at 37°C during 45mn then at 80°C during 20 mn

Ligation:
  • Steril H2O: 9µL
  • Insert (J23115):6µL
  • Vector (E0240): 2µL
  • 10X T4 DNA ligase reaction buffer: 2µL

Mix was incubated at 10mn at room temperature then at 80°C during 20mn before the transformation in DH5a chimiocompetent with 3µL of the ligation product with the same protocol
Aug 26

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