Spectral Karyotype (SKY)

 

DAY 1

PREATMENT
01. ___ Mark slides
02. ___ 2XSSC 37 °C for 30 min (Age slide if necessary)
03. ___ 12ul pepsin/40ml 0.01N HCl 37 °C for 5 min (Adjust for different conditions)
04. ___ 1X PBS 5 min
05. ___ 1X PBS 5min
06. ___ PBS/0.05M MgCl2 5min
07. ___ 1% formaldehyde in PBS/MgCl2 10min
08. ___ 1X PBS RT 5 min
09. ___ 70%, 80%, 100% EtOH 3 min each
10. ___ Air dry
11. ___ Check slide under phase-contrast microscope

DENATURATION
12. ___ 70% Formamide/2X SSC at 70 °C 2 min (Adjust for different cell types)
13. ___ Alcohol-series 2 min each, (cold 70%, 80%, 100% EtOH ), �20 °C then air dry

Probes: (from Spectral Imaging)
14. ___ 80°C 7 min and
15. ___ 37 °C 30-60 min

HYBRIDIZATION
Apply 3ul of probe for 12mm circle / 6ul of proble for 18mm circle on cover slip
16. ___ Seal with rubber cement
17. ___ Store for 2 days at 37 °C in a humidified chamber (up-side-down)

DAY 2

18. ___ Peel rubber cement with a tweezer
19. ___ Put slide with cover slip in 2X SSC and shake for 10 min, remove cover slip if needed
20. ___ 3 times 50% Formamide/2X SSC 8 min 43 °C
21. ___ 2 times 2X SSC 8min 43 °C,
22. ___ 4X SSC/0.1% Tween-20, 2min
23. ___ 100ul PNM buffer (at 4°C) 30 min, 37 °C (optional; vial 2-not used in this protocol)
24. ___ 100ul (1ul anti-dig/200ul PNM) 37 °C (vial 3 if applicable), 45 min
25. ___ 3 times, 4XSSC/0.1% Tween-20, 10min
26. ___ 100ul (1ul anti-CY5 +1ul CY5.5/100ul PNM), 37 °C (vial 4 if applicable), 45min
27. ___ 3 times, 4X SSC/0.1% Tween-20, 10min
28. ___ 10ul DAPI on cover slip

----------------------------------------------------------------------------------------------------------------------------

COMPONENTS

1X PBS/MgCl2  (Vol = 500 ml)
25 ml                                        1M MgCl2
475 ml                                      1X PBS

4XS SC/0.1% Tween20 (Vol = 500 ml)
400 ml                                      ddH2O
100 ml                                      20XSSC
500 ul                                       Tween-20

70%Formamide/2XSSC (Vol = 50 ml)
35 ml                                        Formamide
5 ml                                          20X SSC
10 ml                                        ddH2O

50%Formamide/2XSSC (Vol = 150 ml)
75 ml                                        Formamide
15 ml                                        20X SSC
60 ml                                        ddH2O

PNM buffer (adjust to pH 8.0, spin 30 min to clarify, keep in fridge)
5%                                           nonfat dry milk
0.1%                                        NaN3 in pN buffer

PN Buffer :

10 lilters:
Make 10 liters 0.1 M Na2HPO4 (dibasic)
Make 600 ml 0.1 NaH2PO4 (monobasic)
Titrate the dibasic solution (pH > 9) by adding the monobasic solution (pH ~ 4.5) until pH of 8.0 is reached. 
Add NP-40 to 0.05%