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Transformation of pSB1C3/TOD genes

  1. Centrifuge the ligation
  2. Add 6 ul of each ligation reaction to a sterile 1.5ml tube on ice
  3. Thaw the cells on ice
  4. Carefully transfer 50ul of cells to the ligation reaction. Gently flick the tubes and incubate on ice for 20 minutes.
  5. Heat shock the cells for 45 seconds in a 42'C water bath. Do not shake. Immediately return the tubes to ice for 2 minutes.
  6. Add 950 ul of SOC medium (at room temperature) to the ligation reactions. Incubate for 1.5 hours at 37'C on a shaker.
The controls for this experiment are the resistance and viability controls. 
One tube has 50 ul of highly competent cells and SOC medium added to it.

The samples A (TodX), C (TodF), E(ToBG), positive control, and background control were plated on chlorophenicol resistant agar plates, at the volumes of 20ul and 200ul. A 100ul of only highly efficient cells and SOC medium was plated in a chlorophenicol plate and another 100ul was plated on a LB plate.

Isolate plasmid from overnight broth

The plasmids were isolated using the Omega Plasmid Mini Kit 1, and its protocol was followed.

Nanodrop results

SampleVolumeConcentration ng/ul260/280260/230
TodX 139 37.8 2.02 1.72
TodX 243.8 59.1 1.9 1.74
TodF 142 100.4 1.92.02
TodF 240.526.21.921.66
ToBG 145 53.2 1.941.67
ToBG 24721.62.001.43

Double digest of isolated plasmid with SpeI and XbaI

Master mix for 12 reactions

  • 36 ul of cut smart buffer
  • 1.2 ul of XbaI
  • 2.4 of SpeI
  • 44.4 ul of 5mTris Hcl


SampleDNA (ul)Master mix (ul)5mTris Hcl (ul) added to final volume of 30ul
TodX 113.2 9 7.8
TodX 28.5 9 12.5
TodF 18.5 9 19
TodF 2592
ToBG 19.5 9 11.6
ToBG 22390