colony PCR

Hybridization with alpha-satellite oligonucleotide probe

Probe Labeling

oligo (10µM)	1 µl
buffer 5x	4 µl
CoCl₂ (1/5 dilution)	3 µl
dCTP-P³²	2 µl
Terminal Transferase (Roche) 50 U	2 µl
H₂O	8 µl
total	20 µl

sequence: TTG TGA TGT GTG CGT TCA ACT (approx. posit. 40 of the consensus)
incubate 30 min at 37°C
purify with NucTrap Probe columns (Stratagene) (or similar)

Pre-hybridization

SSPE (20x)	12.5 ml
SDS (10%)	5 ml
milk powder solution (10%)	50 µl
H₂O	32.5 ml
total	50 ml

1 hour at 46°C, 10...50 ml

Hybridization

add labeled probe to prehybridization solution and incubate 16 hours at 46°C

High stringency washing

2 times washing in 2x SSPE, 0.1 % SDS at 46°C
2 times washing in 0.2x SSPE, 0.1 % SDS at 46°C
seal membrane in plastic bag and expose at -80°C over night

or ... Low stringency washing

4 times washing in 2x SSPE, 0.1 % SDS at 46°C
seal membrane in plastic bag and expose at -80°C over night

courtesy of Jacques Puechberty