Team:Stockholm/30 September 2010

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Contents

Andreas

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Digestions

  1. pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS
    • Clones 2 & 3
  2. pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS
    • Clones 1 & 2
  3. pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS
    • Clones 2 & 3


  1:2 1:3 2:1 2:2 3:2 3:3
10X FastDigest buffer 2 2 2 2 2 2
DNA (1 μg) 5.2 4.1 2.5 3.3 4 4.1
dH2O 10.8 11.9 13.5 12.7 12 11.9
FD XbaI 1 1 1 1 1 1
FD PstI 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  • Incubation: 37 °C, 1.45
  • Inactivation: 80 °C, 20 min

Ligations

  • Vector: [Dig pEX.RFP X+P
  pEX.1:2 pEX.1:3 pEX.2:1 pEX.2:2 pEX.3:2 pEX.3:3
10X T4 Ligase buffer 2 2 2 2 2 2
Vector DNA 1.5 1.5 1.5 1.5 1.5 1.5
Insert DNA 8 8 8 8 8 8
dH2O 7.5 7.5 7.5 7.5 7.5 7.5
T4 DNA ligase 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

Quick transformation

  • 1 μl ligation mix
  • 50 μl 0.1 M IPTG
    • pEX.1:2 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
    • pEX.1:3 (pEX.nTAT⋅SOD⋅His.RBS.yCCS 2)
    • pEX.2:1 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 1)
    • pEX.2:2 (pEX.nTra10⋅SOD⋅His.RBS.yCCS 2)
    • pEX.3:2 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2)
    • pEX.3:3 (pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3)

Transformation of BL21

Quick transformation

  • 50 μl competent cells
  • 0.5 μl plasmid
    • pEX.nTra10⋅SOD⋅His
    • pEX.nLMWP⋅SOD⋅His

ON cultures

  • 3 ml LB + appropriate antibiotic; 30 °C
    • pEX.nTAT⋅SOD⋅His (Top10; Amp 100)
    • pSB1K3.BBa_J04450 (Top10; Km 50)