18 Quantitative DNA Fiber Mapping
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Materials
- Refrigerated Centrifuge (MP4R, IEC)
Equipment
- Dry bath (Model 2001, Labline Instruments)
- Fluorescence microscope (Axioskop, Zeiss) equipped with 40x and
63 x oil immersion lenses
- Incubator oven (set to 37°C, Precision Scientific)
- Pulsed Field Gel Electrophoresis (PFGE) system (Biorad)
- Shaking Incubators (New Brunswick): 30°C for yeast cell culture, 37°C
for culture of E. coli
- Thermal cycler for in vitro DNA amplification
- Water bath (Model 188, Precision Scientific)
- Digital Imaging system (optional)
- 3-Aminopropyltriethoxy silane (APS, Sigma Chemicals)
Reagents
- ~-Mercaptoethanol (Sigma Chemicals)
- ~-Agarase (New England Biolabs (NEB»
- Agarose (Life Technologies)
- Antibodies against digoxigenin, rhodamine-conjugated, made in sheep
(Roche Molecular Biochemicals), stock solution is 1 mg/ml in PNM,
dilute 1:50 with PNM prior to use. Store at 4°C.
- Antibodies against FITC, made in mouse (DAKO), stock solution is
1 mg/ml in PNM, dilute 1:50 prior to use. Store at 4°C.
- Anti-mouse antibodies, FITC conjugated, made in horse (Vector Labs),
stock solution is 1 mg/ml in PNM, dilute 1:50 prior to use. Store at 4°C.
- Anti-avidin antibodies, biotinylated, made in goat (Vector Labs), stock
solution is 1 mg/ml in PNM, dilute 1:50 prior to use. Store at 4°C.
- Avidin conjugated to AMCA (Vector Labs), stock solution is 2 mg/ml
in PNM, dilute 1:500 prior to use. Store at 4°C.
.
- Blocking reagent: cat. #1096-176 (Roche Molecular Biochemicals)
- Chloroform/isoamyl alcohol: 24:1 vol:vol. (Life Technologies)