Spheroplasts Plates

Day 0: Overnight growth of 96 selected clones

• 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

• 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

Preparation of the extracts

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

Spheroplasts

• 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

• 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