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CGH Protocol
COMPARATIVE GENOMIC HYBRIDIZATION (CGH) PROTOCOL


For CGH, selected slides with normal metaphase chromosomes are used (stock at -20C). Prior to the making of the chromosome slides, slides are coated with silane.

SAVING OF DAPI-IMAGES

-DAPI-staining solution : 100 ml 1xPBS + 2 l DAPI-stocksolution(1mg/ml)
-incubate slides for 3' in the stainingsolution at RT in the dark
-rinse 3x1' with tap water
-air dry slides and mount in antifade medium (35 l Vectashield/slide)
-15 to 20 mitoses are selected and DAPI images are captured using a black and white CCD camera .

SLIDE PRETREATMENT

Prior to slide pretreatment coverslips are gently removed and slides are rinsed for 15' with 94% ethanol at RT . Subsequently slides are air dried.

1)RNase pretreatment
-incubate slides 1 hour at 37C in 100 g/ml RNase A in 2xSSC (pH 7) in moist chamber
Stock RNase A : 10 mg/ml Dilution : 10 l stock + 990 l 2xSSC
-wash 3x5' with 2xSSC (RT)
-dehydratation in ethanol series : 70%,90%,94% for 3' ( RT) on shaker
-air dry slides

2)Pepsin treatment
-incubate 30' at 37C(WWB) in 0.005% pepsin in 0.01 N HCl
Stock pepsin :10% worksolution : 0.005% (50 l stock + 100 ml HCl both pre-warmed at 37C)
-wash in 5' 1xPBS (RT)

3)Postfixation
-wash 5' in postfixation buffer at RT on shaker :
100 ml postfixation-buffer : 10 ml 10xPBS, 10 ml 0.5 M MgCl2, 80 ml bidest
-5' fixation in 4% paraformaldehyde (PFA) at RT on shaker
100 ml 4% PFA : 10 ml 10xPBS, 10 ml MgCl2, 30 ml bidest, 50 ml 8% PFA
-wash in 1xPBS 5' at RT on shaker
-dehydratation in ethanol series : 70%,90%,94% for 3' each at RT on shaker
-air dry slides

PROBE PREPARATION (on ice)

1. 300 ng biotinylated test DNA (stock : 20ng/ml) and 300 ng dig-labeled control DNA (stock 20ng/l) are combined. Add 25COT-I-DNA (=15 l of stock-conc.,1 g/ml)
2. precipitate with Na-acetate (3 M,pH 5.6) : 1/10 of the total volume of the DNA and ice-cold 100% ethanol : 2.5x of the total volume of the DNA
3. chill on ice for 30'
4. centrifuge 30' at 14000 RPM at 4C
5. remove supernatans (with fine-needle in flow-bench)
6. air dry pellet for 15' (dust free)
7. dissolve the pellet in an appropriate volume of 50% formamide/12.5% dextran- sulphate/2xSSCP (10 l/slide)
8. incubate 30' at 37C(WWB) to dissolve the pellet completely
9. denature probes at 70C(WWB) for 5'
10. chill immediately on ice for 3'
11. preannealing of the probes at 37C(WWB) for 30'
12. denature target DNA (slides) with 100 l 70% formamide/2xSSCP for 2.5' under a coverslip on a hot plate at 75C . The denaturation is started during the last 15' of the preannealing of the probe.
13. chill slides in ice-cold 70% ethanol for 2.5' followed by rinses in 90% and 94% ethanol
14. dry slides on a hot plate (40C) and apply 10 l of preannealed probe under coverslip (18x18 mm or 24x24 mm)
15. 3 or 4 days hybridization in moist chamber (60% form./2xSSCP) at 37C in incubator

PROBE DETECTION

PREPARATION OF SOLUTIONS :
-50% formamide/2xSSC pH 7.0 300 ml :
-150 ml 100% formamide
-30 ml 20xSSC
-120 ml bidest
adjust to pH 7.0 with 6 N HCl
-0.1xSSC
These solutions are both pre-warmed at 42C
-2xSSC
-4xSSC/Tween-20 0.05% (0.5 ml Tween-20/1l 4xSSC)

1. Washing

-3x5' with 50% formamide/2xSSC at 42C (WWB)
-5x2' with 0.1xSSC at 42C
-1x5' with 2xSSC at RT on shaker

2.Blocking

-incubate 20' in moist chamber at RT with 100 l of 4xSCC/Tween

3. Washing

-1x5' in 4xSSC/Tween (RT)

4. Detection

Layer 1 :
Neutralite-Avidin-FITC
dilution : 1/200 in 4xSSC/Tween
-centrifuge 2' on 12000 RPM
-incubate with 100 l/slide in moist in dark at 37C for 30'

Wash 3x5' in 4xSSC/Tween at RT on shaker

Layer 2 :
biotinylated goat-anti-Avidin + mouse anti-dig
dilution : 1/100 + 1/500 in 4xSSC/Tween
-centifuge for 2' on 12000 RPM
-incubate with 100 l/slide in moist in dark at 37C for 45'

Wash 3x5' in 4xSSC/Tween at RT on shaker

Layer 3 :
Neutralite-Avidin-FITC + sheep anti-mouse dig
dilution : 1/200 + 1/100 in 4SCC/Tween
-centifuge for 2' on 12000 RPM
-incubate with 100 l/slide in moist in dark at 37C for 45'

Wash 3x5' in 4xSSC/Tween at RT on shaker

Layer 4 :
sheep anti-dig-TRITC
dilution : 1/100 in 4xSSC/Tween
-centifuge for 2' on 12000 RPM
-incubate with 100 l/slide in moist in dark at 37C for 45'

Wash 2x5' in 4xSSC/Tween (RT)

5. Counterstaining with DAPI

-incubate for 3' in DAPI-stainingsolution (100 ml 4xSSC/Tween + 2 l DAPI-stocksolution (1mg/ml)) at RT in the dark
-rinse 3x1'with tap water, air dry and mount in antifade medium (=35 l Vectashield/slide)