User:Jn323/7 August 2012

From 2012.igem.org

Contents

Electroporation

  • Three strains used: BS168, BS1A833, BSPY79
  • Filter-sterilize buffers
Preparation of Cells
  • Check O/N growth
  • Back-Dilution prepared until OD600 reading was between 0.4 and 0.8

Following steps done on ice

  • Place following items on ice (for future use):
  • Washing & electroporation buffers
  • 0.1cm cuvettes
  • DNA sample
  • Aliquot 1mL of grown cells into 1.5mL eppendorf tubes each
  • Wash cells twice in 1x volume of cold washing buffer
  • Spin down each aliquot in microcentrifuge (4000rpm, 10 minutes at 4°C)
  • Remove supernatant immediately
  • Repeat steps a & b
  • Spin down aliquots once more in same conditions
  • Resuspend each pellet in 50uL of cold electroporation buffer with 500ng of DNA sample
  • Transfer each aliquot into 0.1cm cuvettes
Electroporation
  • Wipe sides of cuvettes prior to electroporation
  • Electroporate at following settings:
  • 1.8kV
  • 200Ω
  • 25 uF
  • Immediately add 1mL SOC media
  • Incubate cultures in shaking incubator at 30°C for 1 hour
  • Transfer SOC-cell suspension to clean 1.5mL eppendorf tubes
  • Spin down in table-top centrifuge for 30 seconds
  • Remove 950uL of supernatant to concentrate in 50uL of LB
  • Resuspend and plate with either autoclaved beads
Results

Electroporation Results

Cell Strain TC (ms) V (V)
BS168 1.1 1783
BS168 - Control 1.3 1785
BSPY79 2.1 1789
BSPY79 - Control 1.8 1787
BS1A833 2.2 1785
BS1A833 - Control 2.5 1789

Note:

During electroporation, arching observed (most likely due to too high salt concentrations)

Plates:

Plates showed no growth (most likely due to unsuccessful electroporation due to arching)