Details on probes (for details see Library information):

YAC CEPH Megalibrary: Resistance:AMP 50ug/ml

BAC CEPH library. Vector: pBeloBAC11. Resistance: Cloramphenicol 12.5 ug/ml
end sequencing:

BACs from P.dejong: Vector: pBACe3.6 (11.49 kb). Resistance: Cloramphenicol 12.5 ug/ml
end sequencing (same name different sequence!!):

PAC: Library DeJong RPCI-5. Vector: pCYPAC2N (18.754kb). Resistance: Kanamycin 50ug/ml
end sequencing:



Final volume: 50ul; use approximately 50ng of the supplied DNA.

Prepare a Taq reaction mix containing 200 uM of dNTP, 100 pmol of universal primer, 10 mM Tris-HCl, pH8,3, 1,5mM MgCl2, 50mM KCl, 1,25 units of Taq DNA polymerase.


30 cycles of
94C for 1 min
62C for 1 min
72C for 2 min
Extension 72C for 8 mins

Slide treatment with pepsin (for ageing fresh made slides)top

Rapid ageing of slides

- prepare slides as usual

- 1.5h in oven at 90°C

- pepsin treatment

Slide treatment with pepsin (for ageing fresh made slides)

- incubate for 30' at 37°C in 0.005% pepsin/0.01M HCl (stock)

- wash 1xPBS at RT for 5'

- 5' at RT in buffer for 50ml (5ml 10xPBS : 5ml 0.5M MgCl2 : 40ml H2O)

- 5' in paraformaldehyde 4% at RT for 50ml (5ml 10xPBS : 5ml 0.5M MgCl2 : 15ml

H20 : 25 ml paraformaldehyde 8%)

- wash 1xPBS, 5' at RT

- 3 wash in ethanol series (70%, 90%, and 100%)

- air drying

DNA extraction from YACs (CEPH megalibrary)top

- seed the YAC in 5-10ml of YPD (with ampicillin, 50ug/ml)
- incubate 2 days at 30°C in a stirrer
- fuge 5' a 3500RPM; discard the SN
- suspend in 0.5ml 1M sorbitol-0.1M EDTA (pH7.5); 
- transfer in eppendorf tube
- add 7ul of lyticase  (stock solution: 10.000U/ml).
- incubate at 37°C for 30'
- fuge for 1', discard the SN.
- suspend in 0.5ml of 50mM Tris-HCl (pH7.4) - 20mM EDTA
- add 50ul of SDS 10%; mix
- at 65°C for 30'
- add 0.2ml of 5M KAc; in ice for 60'
- fuge for 5'
- transfer the SN in a eppendorf tube
- add 1 vol isopropanol at RT
- mix; leave at RT per 5'
- fuge  for 10 seconds, discard the SN, let the pellet dry
- suspend in 0.3ml of TE (pH7.4)
- add 15µl of RNAse A 1mg/ml; at 37°C for 30'
- add 30µl of Na Acet., mix
- precipitate with 0.2ml of isopropanol
- fuge briefly; discard the supernatant 
- dry the  pellet in Savant for 5'
- suspend in 0.1-0.3ml TE (pH7.4)
- fuge briefly to eliminate insoluble material
YPD:  (1 liter):       10gr yeast extract
                                20gr peptone
Autoclave for 30'; when the temperature is around 50C  add 
50ml of glucose (40%, sterilized by filtration)

PAC-BAC Miniprep top

NOTE: Resistance: BAC: Cloramphenicol (12.5 ug/ml);    PAC: Kanamycin (50ug/ml)
               LB Medium (1 liter): Bacto yeast extract 5 g; Bacto trypton 10 g; NaCl 10 g; H2O up to 1 Liter. Autoclave.

(We recently shifted to commercial kits, which usually give more pure DNA).

1. Grow bacteria in 10 ml bacterial medium containing the right antibiotic in 50 ml falcon tubes for 16/20 hours.
2. Centrifuge 7' at 4000 rpm.
3. Add 300µl of GTE (Glucose 50mM,Tris 25mM pH=8, EDTA 10Mm)
4. Resuspend pellet completely.
5. Trasfer the cell suspension into 2.2ml eppendorf tubes.
6. Add 600 µl of denaturation solution freshly made (0.2 N NaOH, 1% SDS, room temp); do not vortex, mix by inverting several times, do not lyse for more than 5'. The lysate should appear viscous.
7. Add 500µl of 7.5M Ammonium Acetate; do not vortex, mix immediately by inverting several times; leave on ice for 10'; invert several times during the incubation period.
8. Centrifuge at 13000 rpm for 20'
9. Pour the supernatant into fresh 2.2ml eppendorf tubes; the supernatant is most often not clear and a second centrifuge at 14000 rpm for 10' needs to be carried out.
10. Pour the supernatant into fresh 2.2ml eppendorf tubes.
11. Add 700µl isopropanol; mix by inverting several times.
12. Centrifuge at 14000 rpm for 20'.
13. Discard supernatant; the pellet should be barely visible
14. Wash the pellet with 500µl of 70%EtOH.
15. Centrifuge at 14000 rpm for 5'.
16. Discard the supernatant; don't let the pellet dry.
17. Resuspend the pellet in 100µl of TE by taping the tubes.
18. Treat with RNAse f.c. 100ug/ml: at 37°C for 30'.
19. Precipitate the DNA with 1/10 vol NaAc and 3 vol Ethanol
20. Incubate at -20°C for 20'
21. Fuge for 15' at 14000 rpm; wash with 70% Ethanol
22. Resuspend in TE (appropriate volume)
23. Check on gel; store at 4°C
Before labeling: fuge for 15' at 14.000 rpm

Alu-PCR amplification top

PCR is performed using the following Alu primers (Liu et al., 1993):


150 ng of genomic DNA from the hybrid is amplified in a volume of 50µl.
- 5 min at 95°C;
- 30 cycles as follows:
1 min at 95°C
1 min at 65°C
4 min at 72°C
final extension of 10 min at 72°C.

Check the concentration of amplified products on gel. A good amplification yields about 200ng/ul
Label the products using Nick-translation as described (see below).

Reagents for PCR (store frozen):
- 10x dNTPs mix 2mM each, pH7.
- 10x reaction buffer (usually comes with the Taq).

Probe labelling with Biotin by nick-translation top

Indirected labelling (Biotin)

1. Add to a microfuge tube, on ice:
- 1µg of DNA
- 5µl 10x nick translation buffer*
- 5µl dNTPs mix*
- 2.5µl biotin mix*
- 5µl 0.1M beta-mercaptoethanol
- dilute (immediately before use) 1µl of DNAseI (2U/µl) in 1ml distilled water
- add 10µl to the nick-translation mix: the DNAseI should be calibrated to give fragments of 100-500bp
- 5U DNA polymerase I
- distilled sterile water to 50µl


Direct labeling (Cy3)

1. Add to a microfuge tube, on ice:
- 1µg of DNA
- 5µl buffer 10x
- 1µl dACG (stock: 0.5mM)
- 0.5µl dUTP-Cy3 (Amersham, stock: 1mM)
- 5µl 0.1M beta-mercaptoethanol
- 0.5µl DNA pol.I (10U/µl)
- dilute (immediately before use) 1µl of DNAseI (2U/µl) in 1ml distilled water
- add 10µl to the nick-tranlation mix: the DNAseI should be calibrated to give
fragments of 100-500bp
- H2O --> a 50µl


Direct labeling (FluorX)

1. Add to a microfuge tube, on ice:
- 1µg of DNA
- 5µl buffer 10x
- 3µl dAGT (stock 0.5 mM)
- 1.5µl dCTP-FluorX (Amersham)
- 5µl 0.1M beta-mercaptoethanol
- 0.5µl DNA pol.I (10U/µl)
- dilute (immediately before use) 1µl of DNAseI (2U/µl) in 1ml distilled water
- add 10µl to the nick-tranlation mix: the DNAseI should be calibrated to give
fragments of 100-500bp
- H2O --> a 50µl
2. Incubate at 15°C for 2h
3. Place at 4°C until it is checked on gel.
4. Take 5µl aliquot of each sample, add 5µl 2x loading buffer. Run on 1% agarose gel. Load also a suitable MW marker (as pCMVb-HaeIII). Inspect the gel on UV transilluminator: fragments should be between 100 and 500bp.
If fragments are larger, add additional DNAseI to the samples, incubate for additional 10' and check again.
5. Stop reaction adding 4µl 0.5M EDTA (not necessary if on ice).

* nick translation buffer (10x buffer):
0.5M Tris-HCl pH 7.8-8
50mM MgCl2
0.5mg/ml BSA

* dNTPs mix:
0.5mM dTTP
0.5mM dCTP
0.5mM dGTP

* biotin-16-dATP mix
0.5mM dATP
0.5mM bio-16-dATP (Boehringer Mannheim)

Fluorescence in situ hybridization (FISH) top

Slide denaturation

1.Prepare 200µl/slide of denaturing solution (70% deionized formamide/2xSSC)
2.Pre-warm slides at 60°C on a thermoblock plate.
3.Put 200µl of denaturation solution on each slide and incubate for exactly 2 min at 80°C on a thermoblock plate.
4.Dehydrate slides in 70, 90 and 100% ethanol, 3m each time (70% ethanol at -20°C).
5.Dry slides after dehydratation.

Probe denaturation

1.Precipitate labelled DNA (see note) with 10µg human Cot-1 DNA (not for repeated sequences), 3µg salmon sperm DNA, 1/10 vol 3M Na acetate and 3 vol cold (-20°C) ethanol. Leave at -80°C for 15 minutes. Spin 15' (14,000 rpm) at 4°C. Dry completely the pellet on a Savant fuge for few minutes
2.Prepare hybridization mix (10 µl per slide). Add to a test tube: 5µl of deionized formamide, 2µl dextran sulphate (50% in distilled water, autoclaved), 2µl distilled water and 1µl 20xSSC. If more slides have to be hybridized, a master mix can be prepared.
3.Resuspend pellet in 10µl hybridization mix, by vortexing accurately for 10 min;
4.Denature DNA mix at 80°C for 8 min; transfer to 37°C for 20 min to make preannealing (not for repeated sequences). Place on ice until used.
NOTE: the amount of probe depends on the type of probe: 20-50 ng for repetitive DNA; 500-600 ng for painting probes, YACs, PACs, BACs, cosmids and plasmids;1000 ng for very small probes (es. Plasmids, cDNAs).


1.Apply 10µl hybridization mix to denatured slides, avoiding air bubbles
2.Cover with 24x24mm clean coverslip; seal with rubber cement
3.Incubate in a moist chamber at 37°C overnight

Post-hybridization washing and detection

Do not allow slides to dry at any passages! All washings are done in Choplin jar.

1.Remove coverslips and wash 3 times for 5min in prewarmed solution (50% form. / 2xSSC) in a Choplin jar in a shaking waterbath at 42°C (we usually skeep this first washing)
2.Wash 3 times for 5' in prewarmed 0.1xSSC in waterbath at 60°C, then go to step 6 if direct labeling has been performed
3.Apply 200µl of blocking solution per slide (3% BSA/4xSSC/0.1 Tween 20 ); cover with 24x50mm coverslip; transfer the slides in a moist chamber; incubate for 30' at 37°C.
4.Dilute stock solution of avidin-Cy3 (1:300) in detection buffer (1%BSA/1xSSC0/0.1 Tween 20) (Cy3 is from Amersham; Cy3 is stronger and more stable than other fluorochromes; use the same filter for rhodamine, or a specific one). Let coverslips slide off; apply 200µl detection solution per slide. Cover with 24x50mm coverlips. Transfer the slides in a dark moist chamber. Incubate at 37°C for 30 min.
5.Remove the coverslips; rinse the slides 3 times for 5 min in pre-warmed washing solution (4xSSC / 0.1 Tween 20) in waterbath at 42°C.
6.Counterstain with DAPI (200ng/ml in 2xSSC)
7.Apply 30µl of antifade-mounting medium* and cover with 24x50mm coverslip; slides can be stored for weeks in the dark at 4°C.


* Antifade-mounting medium (for 10ml):
- 0.233g of DABCO (1,4-diazabicyclo-(2.2.2)octane, Sigma)
- 800µl H2O
- 200µl 1M Tris-HCl
- 9ml glycerol

References top

Archidiacono N, Antonacci R, Forabosco A, Rocchi M: Preparation of human chromosome painting probes from somatic cell hybrids. In: Methods in Molecular Biology, Vol. XX: In situ Hybridization Protocols. Choo KHA, ed. Totowa, NJ: Humana Press Inc., 1-13 (1994)

Archidiacono N, Marzella R, Finelli P, Antonacci R, Jones C, Rocchi M: Characterization of chimpanzee-hamster hybrids by chromosome painting. Somatic. Cell. and Mol. Genet. 20:439-442 (1994)

Antonacci R, Marzella R, Finelli P, Lonoce A, Forabosco A, Archidiacono N, Rocchi M: A panel of subchromosomal painting libraries representing over 300 regions of the human genome. Cytogenet. Cell. Genet. 68:25-32 (1995)

Liu P, Siciliano J, Seong D, Craig J, Zhao Y, de Jong PJ, Siciliano MJ: Dual Alu PCR primers and conditions for isolation of human chromosome painting probes from hybrid cell. Cancer Genet Cytogenet 65:93-99 (1993).

Muller S, Koehler U, Wienberg J, Marzella R, Finelli P, Antonacci R, Rocchi M, Archidiacono N: Comparative fluorescence in situ hybridization mapping of primate chromosomes with Alu-PCR generated probes from human/rodent somatic cell hybrids. Chrom. Res. 4:38-42 (1996)

Spurr NK, Bashir R, Bushby K, Cox A, Cox S, Hildebrandt F, Hill N, Kao F-T, Krols L, Marzella R, Miller N, Nothwang HG, Rocchi M, Sarfarazi M, Stratakis CA, Wallgren-Pettersson C, Naylor S: Report of the four international workshop on human chromosome 2 mapping 1996. Cytogenet. Cell. Genet. 73:255-273 (1996)

Rocchi M, Antonacci R, Marzella R, Finelli P, Cassano C, Lonoce A, Cino C, Forabosco A, Archidiacono N: Subchromosomal painting libraries (SCPLs) from somatic cell hybrids. Chromosomes Today Vol. 12 in press (1996)

Gianfrancesco F, Esposito T, Ruini L, Houlgatte R, Nagaraja R, D'Esposito M, Rocchi M, Auffray C, Schlessinger D, D'Urso M, Forabosco F: Mapping of 59 EST gene markers in 31 intervals spanning the human X chromosome. Gene in press

Marzella R, Viggiano L, Ricco A, Tanzariello A, Fratello A, Archidiacono N, Rocchi M: A panel of radiation hybrids and YAC clones specific for chromosome 5. (These hybrids have been characterized also by STSs) . Submitted for publication.

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