Spheroplasts Plates

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Day 0: Overnight growth of 96 selected clones[edit]

  • Starting from rearraying list, perform an overnight culture of 96 clones in 1 ml of LB with antibiotics to taste.
  • To avoid any mistakes, take a full autoclaved box of 10 microliters tips, and leave tip in deep well, this way you will know which well has been done. Do not forget a positive control in position 96.
  • Grow overnight in HiGro at 37 degC with 300 rpm of shaking. Airpore tape sheet will close the deep well allowing aeration, and providing any contamination.

Day1: Induction in 24 well blocks[edit]

  • Prepare 4 24 well blocks with 4 ml of TB buffered with the phosphate buffer with antibiotics to taste in each wells. Use a dispenser for convenience.
  • Transfer 40 uL of the saturated preculture.
  • Let the 24 well plates at 37C until it reaches OD600 = 0.6, usually this is 3 or 4 hours.
  • Check the OD by measuring it in 2 wells per plates (1 in the periphery and 1 in the center of the plate)
  • Then induce the cells using the appropriate inductor and transfer the plate at 25C
  • (You can try 20C or 37C if you were not satisfied by the result at 25C)
  • Let the plates ON

Day 2: Preparation of the extracts and purification[edit]

Preparation of the extracts[edit]

  • Stop the culture
  • Centrifuge the plates using the appropriate rotor at 3700 rpm during 10min at 4C.

Spheroplasts[edit]

  • Discard the supernatant in a bottle and resuspend the pellet with
  • Tris pH 8 @ 20mM, NaCl 250mM, Sucrose 20% and lysozyme 1mg/mL. Resuspension performed in 4 milliliters per well.
  • Use thermomixer confort at 4OC and set it to 500 rpm to resuspend pellets. Incubate for 5 minutes per plate. If resuspension was not efficient use a pipet to help resuspending. Finally incubate for 30’ on a rocking platform in cold room.
  • Centrifuge plates 3700 rpm during 10min at 4C. Discard supernatant by putting plate upside down.
  • TAKE CARE TO NOT LEAVE ANY TRACE OF SUCROSE, WHILE LYSING CELLS SUCROSE WOULD INHIBIT THIS STEP.
  • Incubate the plates in freezer for 20 minutes to burst cells by one cycle of freeze/thaw.


Lysis[edit]

  • Lysis buffer:Tris pH 7.5 10mM, Brij 0.5%, Benzonase d=1000, Inhibitors cocktails without EDTA, d=1000. Resuspension performed in 700 microliters per well.
  • Use thermomixer confort at 4OC and set it to 500 rpm to resuspend pellets. Incubate for 5 minutes per plate. If resuspension was not efficient use a pipet to help resuspending. Finally incubate for 20’ on a rocking platform in cold room. Then leave plates on ice.
  • During this incubation, dispense 60 microliters of NI NTA resin in each well of receiver plate TECAN.
  • Run TECAN program: Chicken version 2