Clone preparation

DNA purification

DNA sonication

DNA labeling

DOP PCR primers: 5'-CCG ACT GCA GNN NNN NAT GTG G-3', dissolved in distilled water or TE (100 |M) - store at -20°C

dNTP mix (dATP, dCTP, dGTP, dTTP; 100 mM) - store at -20°C

Magnesium chloride (100 mM)

Taq 2000 DNA polymerase (5 U/|l, Stratagene) - store at -20°C

10 x PCR buffer (supplied with Taq DNA polymerase) - store at -20°C

BAC DNA sample

Spotting buffer: 30% dimethyl sulfoxide (DMSO)

QIAamp kit containing lysis buffer AL, wash buffer AW1, wash buffer AW2 and AE elution buffer (QIAgen)

Ethanol (100%)

DNA samples

Distilled water

DNA samples

Distilled water

Bioprime labeling kit containing 2.5 x Random Primed and Klenow polymerase (Amersham Biosciences)

dNTP mix containing 2 mM of the nucleotides dGTP, dCTP, dATP and 1mM of dTTP, 1 mM EDTA pH 8.0 and 10 mM Tris pH 8.0

Cy3-dUTP (Amersham Biosciences)

Cy5-dUTP (Amersham Biosciences)

Purification of labeled DNA Labeled DNA samples

QIAquick kit containing PB buffer, PE buffer and EB elution buffer (QIAgen)

DNA precipitation Purified labeled DNA

Human COT1-DNA (1 |g/|l, Roche) 3 M NaAc pH 5.2 100% ethanol

Probe preparation Precipitated DNA (test and reference)

Distilled water

Yeast tRNA (100 mg/ml, Invitrogen) 20% SDS

Slide pre-treatment

Posthybridization procedure

Gel electrophoresis

Additional reagents and materials:

Mastermix containing 50% formamide, 10% dextran sulfate, 2 x SSC and 4% SDS

Distilled water

20 x SSC

1 M Tris pH 9.0 10% SDS Ethanolamine Formamide

Filter sterile 20 x SSC pH 5.0

Nonidet P-40

Distilled water

0.2 M Na2HPO4

0.2 M NaH2PO4


100 bp marker

Multiscreen plate (Millipore)

50-ml falcon tube

0.5-ml reaction tube, amber

1.5-ml reaction tube

Coplin jar

250-ml beaker 1-l beaker Forceps Bunsen burner

QIAamp DNA purification kit (QIAgen) QIAquick PCR purification kit (QIAgen) CMT-GAPS coated slides (Corning) Exicator


Arrayer (Omnigrid 100, Genomic Solutions) UV Stratalinker Thermal cycler Speedvac

Gel electrophoresis system Power supply

Sonicator (Soniprep; Soniprep150)

Hybridization station (GeneTAC, Genomic Solutions)


Scanner with Cy3 and Cy5 lasers (Affymetrix 428 scanner, Affymetrix)

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