Day 1 Metaphase preparation (35mm plate of ES cells)
1. Feed ES cells at least 1 hour before adding colcemid.
2. Add colcemid to 0.01mg/ml (25ml of 2mg/ml in PBS), swirl to mix.
3. Incubate for 1.5 hr at 37oC.
4. Rinse with PBS, trypsinize cells with 0.2ml 10X trypsin.
5. Add ES media to quench and spin down at 1000rpm for 5 min.
6. Discard supernatent and flick pellet to resuspend.
7. Add 0.075M Kcl. resuspend well and allow to sit at rt for 15 min.
8. Prepare fresh ice cold fixative; 3:1 Methanol: Glacial Acetic Acid.
9. Add a few drops of freshly prepared fixative. Invert to mix.
10. Spin, 1000rpm, 5min..
11. Add 5ml cold fixative, mix well by pipetting. Rt, 10min.
12. Spin, 1000rpm, 5min and repeat twice.
13. Repeat wash 2X with fixative.
14. Resuspend in 1-2ml, -20oC, O/N.
15. Soak glass slides in glacial acetic acid, O/N.
Day 2 Dropping Slides
1. Add fresh prepared fixative to 10ml, spin, 1000rpm, 5min.
2. Resuspend in fixative. (It is important to keep the cultures fairly diluted for good spreads)
3. Wash slides in deionized water, 100% EtOH and deionized water, air dry.
4. Hold slide at an angle and place 2-3 drops of cell suspension on slide,
Allow the cells to spread.
5. Allow to air dry and let the slides age overnight at room temperature
6. Slides can be stained with Giemsa, 5-7 min for chromosome count.
Day 3 Denaturing and Hybridization
1. Prewarm the airtight humidity chamber at 37oC
2. Incubate the denaturing solution at 70oC, 30min
3. Mark areas for hybridization.
4. To remove cell debris, immerse slides in
2X SSC 2min
70% EtOH 2min
85% EtOH 2min
100% EtOH 2min
5. Allow to air dry.
6. Immerse slides in 70oC denaturation solution, 2min
7. Immediately, 70% EtOH, 2min, rt.
8. 85% EtOH, 2min, rt.
9. 100% EtOH, 2min, rt.
10. Drain excess and allow to air dry.
11. Warm hybridization buffer to rt.
12. Per slide, use 7l hybridization buffer + 3l probe, vortex, spin down.
11. Warm slides in 37oC incubator, 10min.
12. Denature probe in 70oC, 5min, ice.
13. Add 10l probe to marked area, place coverslip on top.
14. Keep slide in humidity chamber, 37oC, in the dark, O/N.
Day 4 Washing
1. Formamide x 3, 2X SSC, 2X SSC with Nonidet, 45oC, 30min.
2. Remove slide from 37oC, remove coverslip.
3. Wash:
formamide 1 5 min
formamide 2 5 min
formamide 3 5 min
2X SSC 5 min
2X SSC/Nonidet 5 min
4. Drain excess, airdry in the dark.
5. Apply 10l counterstain, place coverslip to spread.
6. Seal edges of coverslip with nail polish.
7. Store slides in the dark, 4oC.
SOLUTIONS USED FOR Y.ES KIT
Denaturing buffer:Formamide: 49ml, 20X SSC: 7ml, H2O: 14ml, pH 7-8 =70ml
70% EtOH: EtOH: 42ml, H2O: 18ml , =60ml
85% EtOH: EtOH: 51ml, H2O: 9ml, =60ml
4oC, discard after 1 week
Formamide Wash:Formamide: 105ml, 20X SSC: 21ml, pH 7-8 =210ml
2X SSC: 20XSSC: 6ml, H2O: 54ml, H2O: 84ml, =60ml
2XSSC/0.1%Nonidet: 20XSSC: 6ml,Nonidet P40: 6ml, H2O: 54ml, =60ml
4oC, discard after 1 week