YEAST CHROMATIN SPREADS
- Spin down 5mL cells OD ~0.4-1.0; Resuspend in 1mL sol1 -->keep ice
- Spin down cells (10K, 10") and resuspend plt 300ul sol 1. Add 6 ul 1M DTT.
- 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.
- Transfer spheroplasts into 4mL cold sol2 (15mL falcon) -30'/ice. spin down 8', 800rpm.
- Resuspend plt (aspirate SN gently!) in 200 ul sol2. May store 4oC O/N.
- 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:
- Place slides in 1X PBS --10'
- Add 300 ul 10% BSA/PBS --10' humid chamber; pour off but do not let dry
- 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)
- Wash in 10' in 1xPBS
- 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.
- Wash 10' in 1X PBS
- 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
|
