|
|
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:
T7 GTAAAACGACGGCCAGT
SP6
AGCGGATAACAATTTCACACAGG
----------------------------------------------------------
BACs
from P.dejong: Vector: pBACe3.6 (11.49 kb).
Resistance:
Cloramphenicol 12.5 ug/ml
end sequencing (same name different sequence!!):
T7
CGGTCGAGCTTGACATTGTAG
SP6
GATCCTCCCGAATTGACTAGTG
----------------------------------------------------------
PAC:
Library DeJong RPCI-5. Vector: pCYPAC2N (18.754kb).
Resistance:
Kanamycin 50ug/ml
end sequencing:
T7
CGGTCGAGCTTGACATTGTAG
SP6 GATCCTCCCGAATTGACTAGTG
Primers: CCGACTCGAGNNNNNNATGTGG
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.
Cycles:
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)
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
- 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)
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
PCR is performed using the following Alu primers (Liu et al., 1993):
5' GGATT ACAGG YRTGA GCCA
3'
5' RCCAY TGCAC TCCAG CCTG 3' (Y=C/T; R=A/G),
150 ng of
genomic DNA from the hybrid is amplified in a volume
of 50µl.
Cycles:
- 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).
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)
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).
Hybridization
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
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.
go to
the top of the page
|
|
|
|
|
|
|
|