Centre for Microarray Resources, Dep’t of Pathology, University of Cambridge
Protocol: aCGH hybridisation (manual)
Author: Anthony Brown
Updated: 01/11/06
aCGH hybridization (manual)
Materials
 aCGH arrays printed on Codelink slides
 Combined labeled sample and reference DNAs, precipitated and stored under ethanol (see aCGH
labeling)
 Human COT-1 DNA (Invitrogen 15279011)
 Herring sperm DNA (Sigma D7290)
 Hybridisation buffer (50% formamide, 2x SSC, 10 mM Tris-HCl pH 7.5, 0.1% (v/v) Tween 20, 10%
dextran sulfate), stored aliquoted at -20 °C
 Hybri-Slips: 22x22 mm (Sigma-Aldrich Z365904-100EA) for 16 block arrays; 22x40 mm (SigmaAldrich Z365912-100EA) for 32 block arrays
 Hybridisation box: a plastic 50 slide box with slots on which the slides can be supported above the
bottom of the box. Alternatively a small airtight Tupperware box containing a supporting surface for
the slides, may be used. The base of the box should be lined with 2 kimwipes, freshly wetted with
50% formamide/2x SSC (failure to use fresh 50% formamide/2x SSC may result in high background
because of evaporation of the hybridization solution)
 25 slide square glass staining dish (120mm x 120mm x 75mm) with stainless steel rack
 Vertical glass staining jar
 1x PBS/0.05% Tween 20
 50% formamide/2x SSC
 1x PBS
 Centrifuge drying
Cautions
 When preparing hybridization buffer, use freshly prepared 50% dextran sulfate solution. Store
hybridization buffer aliquoted at -20 °C
 Blocking and Hybridisation Solutions are very viscous. Pipette them slowly to reduce waste lost to
the pipette tip
 Take care not to introduce bubbles to Blocking and Hybridisation Solutions when pipetting them onto
Hybri-Slips. If a bubble is introduced, aspirate back into the pipette tip, return to the reaction tube and
pulse-centrifuge to remove.
 Do not apply the array to Blocking or Hybridisation Solution containing bubbles. If bubbles are
introduced between the array and Hybri-Slip, do not press the cover glass to remove – doing so will
irreversibly reduce the hybridization volume under the Hybri-Slip
 Do not allow array surfaces to dry when transferring between slide washes
 Where practical, protect Cy-dyes from bright light. Use dark tube racks and cover reactions during
incubation
Reagent preparation
Hybridisation Solution (2.5 µg/µl human COT-1 and 15 µg/µl herring sperm DNA in hybridization
buffer)
1. In a 1.5 ml reaction tube, mix 250 µl of human COT-1 DNA and 150 µl of herring sperm DNA
2. Add 40 µl of 3 M sodium acetate pH 5.5 and 1 ml of absolute ethanol, then incubate at either -20 °C
for 2 hours, -80 °C for 30 minutes or on dry ice for 15 minutes to precipitate the DNA
Centre for Microarray Resources, Dep’t of Pathology, University of Cambridge
Protocol: aCGH hybridisation (manual)
Author: Anthony Brown
Updated: 01/11/06
3. Pellet at full speed in a benchtop microcentrifuge for 15 minutes
4. Decant and discard the supernatant, and replace with 1 ml of 70% ethanol. Mix gently by inverting
the tube
5. Centrifuge at full speed in a benchtop microcentrifuge for 5 minutes
6. Decant and discard the supernatant, and keeping the tube inverted, gently tap the top of tube on a
folded tissue to remove remaining droplets. Take care not to dislodge the pellet from the tube base
7. Immediately pulse-spin the tube in a benchtop microcentrifuge to collect any remaining ethanol in the
base of the tube. Carefully remove the remaining ethanol from the tube with a P10 tip, taking care
not to touch the pellet
8. Allow the pellet to dry at room temperature for 5 minutes
9. Add 100 µl of hybridisation buffer to the pellet. Incubate the tube at 75 °C for 10 minutes, flicking the
tube vigorously every few minutes to dissolve the pellet. Do not proceed until the pellet has dissolved
completely. Vortex if necessary. Store at -20 °C until required
Slide Blocking Solution (15 µg/µl herring sperm DNA in hybridization buffer)
1. Place 400 µl of herring sperm DNA in a 1.5 ml reaction tube
2. Follow steps 2 – 8 (above, Hybridisation Solution)
3. Add 266 µl of hybridisation buffer to the pellet. Incubate the tube at 75 °C for 10 minutes, flicking the
tube vigorously every couple of minutes to dissolve the pellet. Do not proceed until the pellet has
dissolved completely. Vortex if necessary. Store at -20 °C until required
Method
Slide blocking
1. Prepare a hybridization chamber by placing a layer of tissue in the base of a 50 position plastic slide
box. In a fume hood, wet the tissue with 50% formamide/2xSSC, pouring away any excess
2. Incubate Slide Blocking Solution to 75 °C for 10 minutes to denature the herring sperm DNA
3. Remove the films from both sides of a Hybri-Slip, and place on the top edge of an empty clean tip
box
4. Pulse-spin the denatured Slide Blocking Solution in a benchtop microcentrifuge and apply 30 µl or
60 µl, respectively, to the centre of the 22x22 mm or 22x40 mm HybriSlip, taking care not to
introduce any bubbles
5. Lower the array gently onto the Hybri-Slip. When fixed, gently invert the slide. Check the position of
the Hybri-Slip with an array template, and adjust carefully with clean forceps, if necessary, until the
array is covered
6. Place in a hybridisation box containing tissue thoroughly wetted with 2xSSC/50% formamide.
Incubate in a waterbath at 37 °C for 2 hours
Prehybridisation
7. Pellet the labeled DNA samples at full speed in a benchtop microcentrifuge for 15 minutes
8. Decant and discard the supernatant, add 250 µl of 70% ethanol, mix by inversion and spin at full
speed in a benchtop microcentrifuge for 5 minutes
9. Decant and discard the supernatant, and keeping the tube inverted, gently tap the top of tube on a
folded tissue to remove remaining droplets. Take care not to dislodge the pellet from the tube base
10. Immediately pulse-spin the tube in a benchtop microcentrifuge to collect any remaining ethanol in the
base of the tube. Carefully remove the remaining ethanol from the tube with a P10 tip, taking care
not to touch the pellet
11. Allow the pellet to dry at room temperature for 5 minutes
12. To the pellet, add 25 µl or 50 µl of Hybridisation Solution, for 16- or 32-pin arrays, respectively.
Incubate the tube at 75 °C for 10 minutes, flicking the tube vigorously every few minutes to dissolve
the pellet. Do not proceed until the pellet has dissolved completely. Vortex if necessary
13. Denature at 75 °C for 10 minutes, pulse-spin in a benchtop microcentrifuge, and incubate at 37 °C
for 2 hours.
Centre for Microarray Resources, Dep’t of Pathology, University of Cambridge
Protocol: aCGH hybridisation (manual)
Author: Anthony Brown
Updated: 01/11/06
Hybridisation
14. Remove the coverslip from the blocked array by repeated dunking in 1x PBS in a square glass
staining dish. Transfer quickly to a vertical glass staining jar of 1x PBS and place on a rocking
platform at full speed for 5 minutes
15. Transfer to a slot of a 50 position plastic slide box lined with a dry kimwipe and dry by centrifugation
at 1000 rpm for 3 minutes in a 96-well spin-out plate rotor
16. Remove the films from both sides of a Hybri-Slip, and place on the top edge of an empty clean tip
box
17. Pulse-spin the Hybridisation Solution in a benchtop microcentrifuge and apply 22 µl or 45 µl,
respectively, to the centre of the 22x22 mm or 22x40 mm Hybri-Slip, taking care not to introduce any
bubbles
18. Lower the array gently onto the Hybri-Slip. When fixed, gently invert the slide. Check the position of
the Hybri-Slip with an array template, and adjust carefully with clean forceps, if necessary, until the
array is covered
19. Place in a hybridisation box containing tissue thoroughly wetted with 2xSSC/50% formamide.
Incubate in a waterbath at 37 °C for 21-24 hours
Washing
20. Remove the coverslip from the hybridised array by repeated dunking in 1x PBS/0.05% Tween 20
21. Transfer quickly to the outer positions of a stainless steel rack in a square glass staining dish
containing 400 ml of 1x PBS/0.05% Tween 20. Place on a magnetic stirrer stir, add a stir bar and stir
in the dark for 10 minutes (cover with foil if necessary). A gentle vortex should be visible on the
surface of the solution
22. Transfer the rack quickly to a square glass staining dish containing 400 ml of fresh 1x PBS/0.05%
Tween 20. Place on a magnetic stirrer stir, add a stir bar and stir in the dark for 10 minutes. A gentle
vortex should be visible on the surface of the solution
23. Repeat step 22
24. Transfer to a prewarmed vertical staining dish containing prewarmed 2x SSC/50% formamide and
incubate on a fast rocker at 42 °C in the dark for 30 minutes.
25. Repeat step 22
26. Transfer the rack quickly to a square glass staining dish containing 400 ml of fresh 1x PBS (without
Tween 20). Place on a magnetic stirrer stir, add a stir bar and stir in the dark for 10 minutes. A
gentle vortex should be visible on the surface of the solution
27. Repeat step 26
28. Transfer to a slot of a 50 position plastic slide box lined with a dry kimwipe and dry by centrifugation
at 1000 rpm for 3 minutes in a 96-well spin-out plate rotor