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Revision as of 10:58, 10 February 2008 by Tassos (New page: ==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...)
Day 0: Overnight growth of 96 selected clones[edit | edit source]
- 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 | edit source]
- 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 | edit source]
Preparation of the extracts[edit | edit source]
- Stop the culture
- Centrifuge the plates using the appropriate rotor at 3700 rpm during 10min at 4C.
Spheroplasts[edit | edit source]
- 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 | edit source]
- 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