Team:Washington/Protocols/plate expression

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#Day 1:  OVERNIGHTS
#Day 1:  OVERNIGHTS
##Pick a single colony from plate and inoculate 0.5ml of room temp LB/Kan (NOT TB) in STERILE deep well plate (Round bottom 2 mL Deepwell plate; e.g. Cat No # 7701-5205)
##Pick a single colony from plate and inoculate 0.5ml of room temp LB/Kan (NOT TB) in STERILE deep well plate (Round bottom 2 mL Deepwell plate; e.g. Cat No # 7701-5205)

Revision as of 03:59, 11 September 2011

  1. Day 1: OVERNIGHTS
    1. Pick a single colony from plate and inoculate 0.5ml of room temp LB/Kan (NOT TB) in STERILE deep well plate (Round bottom 2 mL Deepwell plate; e.g. Cat No # 7701-5205)
      1. Generally we have been picking and growing up 4 individual colonies/mutation, so we have quadruplicate data on each mutation. Plate assays have a lot of variability and each colony is not guaranteed to have the mutation, so quadruplicate assaying helps significantly.
    2. Cover with breathable seal
    3. Shake at 37deg 16-24hrs in warm room plate shaker (Heidolph Brinkmann Titramax 1000 @ 1200rpm) to ensure they are all fully grown
  2. Day 2: EXPRESSION
    1. Vortex the overnights at 1500rpm for 30sec to ensure to mix any cells that have settled to the bottom into the media.
      1. Make glycerol stocks if first time doing this set: 110uL cells + 110uL 20% glycerol. Store in -80.
    2. Inoculate a fresh STERILE plate of 1.0ml of TB/Kan in a deep well plate with 20uL of overnight cultures
    3. Cover with breathable seal
    4. Grow at 37deg as above for 3 hours (target OD approx 0.3-0.8)
    5. Get IPTG out of freezer to defrost for 3 hours - don't let it defrost longer than 3 hours
    6. Add IPTG to 0.5mM (50uL of 10mM IPTG) using the repeater
    7. Transfer plate to 18deg shaker (Heidolph Brinkmann Titramax+Incubator 1000 @ 1200rpm in cold room, preheat to 18) and shake at 1200rpm for 24-30hrs.
  3. Day 3: STORE
    1. Spin down cells at 4000rpm for 20min
    2. Pour off supernatant
    3. Store cells in -20 until ready for purification
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