Team:Stockholm/25 September 2010

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Contents

Andreas

Cloning and assembly

Digestion

Further digestions in addition to the ones performed 24/9.

  pK.N-TAT⋅ SH (4) X+P pK.N-LMWP⋅ SH (1) X+P pK.N-LMWP⋅ SH (1) S+P
10X FastDigest buffer 2 2 2
DNA (1 μg) 8 4 4
dH2O 8 12 12
FD XbaI 1 1 0
FD SpeI 0 0 1
FD PstI 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 37 °C, 1 h
  • Inactivation: 80 °C, 30 min (including digestion samples from 24/9)

Ligation

Underlined sequences relevant for assembly

  1 2 3 4 5 6 7 8
10X T4 Ligase buffer 2 2 2 2 2 2 2 2
Vector DNA (50 ng) 1 1 1 1 1 1 1 1
Insert DNA 5 5 5 5 4 4 4 4
dH2O 11 11 11 11 12 12 12 12
T4 DNA ligase 1 1 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  1. pEX.N-LMWP⋅SOD⋅His (K)
    • pSB1K3.N-LMWP⋅SOD⋅His X+P
    • pEX.SOD X+P
  2. pEX.N-TAT⋅SOD⋅His
    • pSB1K3.N-TAT⋅SOD⋅His X+P
    • pEX.SOD X+P
  3. pEX.N-Tra10⋅SOD⋅His
    • pSB1K3.N-Tra10⋅SOD⋅His X+P
    • pEX.SOD X+P
  4. pEX.N-LMWP⋅SOD⋅His (C)
    • pSB1C3.N-LMWP⋅SOD⋅His X+P
    • pEX.SOD X+P
  5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-LMWP⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-TAT⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-Tra10⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS
    • pSB1C3.N-LMWP⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P

Transformation

  • Standard transformation according to protocol.
    • 1 μl ligation mix
    • Amp 100, Cm 25 or Km 50 plates
    • 50 μl IPTG (pEX constructs, Amp 100 plates)

Sequencing preparation

Prepared samples for sequencing to be sent on Monday.

Construct Primer Sample name Sequencing code
(edited 27/9)
pEX.N-TAT⋅SOD⋅His 3 pEXf pEX.nTAT*SH3_pEXf ASB0045 614
pEX.N-TAT⋅SOD⋅His 4 pEXf pEX.nTAT*SH4_pEXf ASB0045 615
pSB1A2.RBS.yCCS 3 VF2 pA.RBS.yCCS3_VF2 ASB0045 603
pSB1A2.RBS.yCCS 4 VF2 pA.RBS.yCCS4_VF2 ASB0045 604
pSB1K3.N-TAT⋅SOD⋅His 4 VF2 pK.nTAT*SH4_VF2 ASB0045 608
pSB1K3.N-TAT⋅SOD⋅His 5 VF2 pK.nTAT*SH5_VF2 ASB0045 609
pSB1K3.N-Tra10⋅SOD⋅His 5 VF2 pK.nTra10*SH5_VF2 ASB0045 610
pSB1K3.N-LMWP⋅SOD⋅His 1 VF2 pK.nLMWP*SH1_VF2 ASB0045 607
pSB1C3.N-LMWP⋅SOD⋅His 1 VF2 pC.nLMWP*SH1_VF2 ASB0045 605
pSB1C3.N-LMWP⋅SOD⋅His 4 VF2 pC.nLMWP*SH4_VF2 ASB0045 606
pEX.SOD pEXf pEX.SOD_pEXf ASB0045 611
pEX.yCCS 5 pEXf pEX.yCCS5_pEXf ASB0045 616
pEX.SOD⋅His pEXf pEX.SH_pEXf ASB0045 612
pEX.His⋅SOD pEXf pEX.HS_pEXf ASB0045 613

BL21 transformation

Transformed BL21 cells with not-yet-verified pEX.N-TAT⋅SOD⋅His plasmid samples 3 & 4

  • Standard transformation according to protocol.
    • 1 μl ligation mix
      • 100 μl cell aliquot divided in 2x50 μl to lower number of colonies after transformation.
    • Amp 100 plates

Preparation of LB agar plates

Made new Cm (25 μg/ml) and Km (50 μg/ml) LB agar plates.

  • 10X Cm 25
  • 10X Km 50
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