16 Comparative Genomic Hybridization
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I. Add PCR/PK Buffer into a O.5-ml PCR tube. Use 60 III for a l-mm
2
Procedures
sample and vary proportionally if the sample is larger or smaller.
2. Use a fresh blade for each region. Scrape the area corresponding to the
marked area on the H&E slide from the methyl green slide. This is done
by taking a small drop of PCR/PK buffer with a #15 blade or a #22
needle, placing the buffer onto the sample area, and scraping tissue
into the center of the drop. When the sample dries enough to be picked
up (don't overdry!), move the scrapings into the PCR tube, overlay with
20 III mineral oil and parafilm close.
3. Incubate 3 days shaking at 120 rpm, 55°C. Add fresh proteinase K (1llg
per 2.5 III original volume) through the oil to the sample twice each day.
4. Remove all parafilm from tubes. Inactivate proteinase K by incubating
10-15 min at 95°C. Remove oil by rolling samples and oil on parafilm
and pipette aqueous DNA into a new tube.
DOP-PCR and labeling for microdissected, Tween extracted DNA samples
- DOP Primer: 5'-CCGACTCGAGNNNNNNATGTGG-3'22mer (Operon) Reagents
- DNA Sample: 1 ng of DNA from fIxed samples prepared as noted above
produces good enough DNA quality for excellent CGH profiles. We
have been able to reproducibly obtain faithful CGH profiles using
as little as 50 pg of DNA from frozen tissue.
- Buffer A
- 2.5 III 600 IlM dNTP (300 IlM fInal concentration)
- 0.5 III 10IlM DOP primer (1 IlM fInal concentration)
- 1.0 III 5x Sequenase Reaction Buffer (200 mM Tris-HCI pH 7.5,
100 mM MgClz
- 250 mM NaCI) (Amersham, Cleveland, OH)
- Buffer B (Taq reaction. Boehringer Mannheim, Germany)
- 16.6 III 600 IlM dNTPs (200 IlM fInal concentration)
- 7.0 III 10 IlM DOP primer (1.4 IlM fInal concentration)
- 5.0 III lOx PCR buffer (Boehringer/Mannheim) (1.5 mM MgClz)
- 2.0 III 25 mM MgClz (1 mM fInal concentration)
- 0.5 III Taq polymerase (2.5 units)
- 13.9 III H 2 0