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VOLKER JUNG and GEORG A. WEBER
- collection buffer (see recipe)
- PCR mix (see recipe) containing biotinylated or fluorochrome-labeled
nucleotides (e.g. Spectrum Orange- or FITC-dUTP)
- ThermoSequenase
- Mayer's hemalaun stain (Merck 1.09249.0500)
- 0.2 ml thin-walled microcentrifuge tubes
- thermal cycler with heated lid
- mineral oil
Collection buffer
- 50 mM Tris Base
- 1 mM EDTA pH 8.0
- 1% Tween 20
- 2 mg Proteinase K/ml buffer
- store at -20°C in 100 ~l aliquots
Hybridization mixture
- 50% Deionized formamide
- 2xSSC
- 10% Dextran sulphate
- 50 mM Na phosphate buffer
- store at -20°C in 500 ~l aliquots
Note: Exceptional care must be taken in preparing reagents for microdissection. Each solution should be prepared with ultrapure water (e.g.
AMPUWA). All plasticware used should be UV irradiated and the
glassware should be autoclaved. It is useful to aliquot all reagents
for one-time use and store them at -lO°e. When preparing solutions,
wear gloves and, ifpossible, conduct all manipulations under a laminar
air flow to minimize the risk of a contamination of extraneous DNA.