Team:Imperial College/Transformation 2

Equipment

• Centrifuge
• Water bath

Reagents and Materials

-SOC broth, per litre: - 10ml

• 2% tryptone (20g)
• 0.5% yeast extract (5g)
• 10mM NaCl (0.5844g)
• 2.5mM KCL (0.186375g)
• 10mM MgCl2 (2.033g)
• 10mM MgSO4
• 20mM glucose (3.6g)

-LB broth, per litre:-110ml

• 1% tryptone,
• 0.5% yeast extract
• 0.5% NaCI

-10% PEG 6000-10ml
-One LB agar plate containing streptinomycin 100ug/ml
-Dry Ice
-1.5ml Eppendorf tubes
-50ml Falcon Tubes

Protocol

Preparation of Competent Cells
Day 1

• Inoculate 10ml of LB media in a 100ml flask from a single colony of B. subtilis on an LB agar plate and grow overnight at 37^oC.

Day 2

• Pre-warm 100ml of LB broth (in a 1 litre flask) at 37^oC. Remove 1ml of this media and measure the OD₆₀₀, keeping this cuvette as will later act as a blank.
• Collect the overnight culture, remove 0.5ml of the overnight culture and add 1ml of LB media in a curvette and measure the O.D.₆₀₀. Remove the remaining 9ml of the overnight culture and add to 100ml of LB broth. Grow this culture at 37^oC with vigorous aeration (200rpm) to an OD₆₀₀ of 1.5.
• Once at an OD₆₀₀ of 1.5 the cells should be aliquoted into 4x50ml Falcon tubes and centrifugation at 4000 rpm for 15 minutes at RT. After this, remove the supernatant and resuspend the pellets in 10ml of ddH₂O. Combine the 4x resuspended pellets so we get 2x50ml falcon tubes and repeat spin. After this remove the supernatant and resuspend in 25ml of ddH₂O and repeat the spin.
• After the last wash the cells are resuspended in 30% polyethyleneglycol (PEG) 6000 to 1% of the original culture volume (So if our original volume was 100ml then we resuspend in 1ml). To do this pipette 1ml of PEG into one pellet and resuspend, then pipette this suspension into the next pellet and resuspend.
• This suspension should then be separated into 100ul samples and pipetted into 1.5ml eppendorf tubes. After pipetting each aliquot place into a tray of dry ice to rapidly freeze samples and store at -80^oC.

Electroporation

• Remove a 100 μl aliquot and thaw on ice. Add 50μl of cells to a fresh eppendorf tube (50μl = 1 condition). To this add the DNA (Concentrations should range from 50ng-500ng) in a total volume of 10μl (Make up to water if necessary).
• Now add to pre-chilled 0.1cm Electroporation gap cuvettes and perform transformation with the following settings:
  • Field Strength - 12kv/cm (1.2kv)
  • Capacitamce - 25µF
  • Resistance - 400 ohms
  • Pulse Length - 8msec
• After transformation add 1ml of SOC broth to the cuvette and pipette into a 15ml falcon tube where a further 1ml of SOC broth should be added.
• Place these tubes into the incubator at 37^oC with gentle shaking for 90 minutes to allow expression of the antibiotic resistance.
• After 90 minutes the cells should be centrifuged at 4000rpm for 15 minutes at room temperature. After spinning the majority of the supernatant should be removed, leaving enough media to allow resuspension ~ typically 100-200μln is ideal.
• This suspension should be aliquoted into the centre of an LB plate containing suitable resistance and streaked out on an LB agar plate.