18 Quantitative DNA Fiber Mapping
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- lOx Taq buffer: 500 mM KCI, 100 mM Tris HCI, pH 8.3,10 mM MgClz.
- TBE (Tris/borate/EDTA) buffer, lOx is 890 mM Tris base, 890 mM
boric acid, 20 mM EDTA.
- TE (Tris/EDTA) buffer, 1x is 10 mM Tris HCI, 1 mM EDTA, pH 7.4, 7.5
or 8.0.
- TE 50 buffer: 10 mM Tris HCI, 50 mM EDTA, pH 7.8.
- Tris HCI [tris(hydroxymethyl)aminomethane]: 1 M, pH 7.5 or 8.0.
Subprotocol 1
Preparation of aminopropyltriethoxysilane (APS) derivatized slides
The derivatization of glass substrates is among the most critical steps of
the procedure. The slides should have the capacity to bind DNA molecules
at one or both ends, but allow the molecules to stretch during the subsequent drying.
Procedure
1. Slide preparation
1. Clean glass slides mechanically by repeated rubbing with wet cheesecloth to remove dust and glass particles.
2. Rinse several times with ultrapure water.
3. Immerse slides in boiling ultrapure water for 10 min.
4. Air dry.
5. Immerse slides in 18 M sulfuric acid for at least 30 min to remove organic residues.
6. Immerse in boiling water for 1-2 min.
7. Air dry and store until further use.