Koshland Web Site/Methods
  1. Spin down 5mL cells OD ~0.4-1.0; Resuspend in 1mL sol1 -->keep ice
  2. Spin down cells (10K, 10") and resuspend plt 300ul sol 1. Add 6 ul 1M DTT.
  3. Add zymolyase (3 mg/ml zymolyase of T20 or T100). Spheroplasting should be done rapidly (~10') as cells are not fixed. Table provides a rough guide for amounts of zymolyase to achieve rapid spheroplasting.
    Temperature amt zymolyase
    23 15 µl
    37 4.2 µl
    Remove aliquot and make 0.1% SDS. Check lysis. Spheroplasting done when 90% cells lyse.
  4. Transfer spheroplasts into 4mL cold sol2 (15mL falcon) -30'/ice. spin down 8', 800rpm.
  5. Resuspend plt (aspirate SN gently!) in 200 ul sol2. May store 4oC O/N.
  6. To pre-cleaned slides** add in rapid succession
    +20 ul cells
    +40 ul fix
    +80 ul 1% liposol
    +80 ul fix

    (using side of a blue pipet tip, draw solution over slide; dry slide 2 hr to O/N in fume hood)

IIF for spreads:

  1. Place slides in 1X PBS --10'
  2. Add 300 ul 10% BSA/PBS --10' humid chamber; pour off but do not let dry
  3. Add 150 ul primary Ab diluted in 10% BSA PBS --2 hr at Rt (Dilute ~1:500 for commercial Mouse anti-HA and Rabbit anti-myc)
  4. Wash in 10' in 1xPBS
  5. Add 150 ul of 2nd Ab in 10% BSA PBS (Aniibody, dilution: Cy3, 1/500; FITC, 1/200). Cover and incubate 1.5 hr at Rt.
  6. Wash 10' in 1X PBS
  7. Shake off extra buffer & use ~ 20 µl DAPI mounting media/coverslip & seal with rubber cement
    SOL1: 0.1M KP pH7.4, 0.5mM MgCl2 + 1.2M sorbitol
    SOL2: 0.1M MES pH6.4, 0.5mM MgCl2, 1mM EDTA +1M sorbitol

    Fix: 4% paraformaldehyde in 3.4% sucrose. Can use up to 6 weeks (see reagents for 20% paraformaldehyde stock)

    **Cleaning slides: boil 10' in 0.01M HCl; rinse w/ EtOH & dry
Comments: Vinny says anti-HA 16B12 gives less background then 12CA5.
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