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From 2012.igem.org

Contents 1 Electroporation 1.1 Preparation of Cells 1.2 Electroporation 1.3 Results

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)