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JosE LUIS FERNANDEZ, VICENTE GOYANES, and JAIME GOSALVEZ
- 0.03 M NaOH
- 1 M NaCl
- Neutralising and lysing solution 1 (pH 7.5), stored at 4°C, used at room
temperature
- 0.4 M Tris HCl
- 0.8 M DTT (Irritant)
- 1% SDS (Harmful if swallowed. Irritant)
- 0.05 M EDTA
- Neutralising and lysing solution 2 (pH 7.5), stored at 4°C, used at room
temperature
- 0.4 M Tris HCl
- 2 M NaCl
- 1% SDS
- Neutralising solution (pH 7.5), stored at 4°C, used at room temperature: 0.4 M Tris HCl
- Isotonic saline solution, stored at 4°C: 0.9% NaCl
- TBE buffer (pH 7.5), stored at room temperature
- 0.09 M Tris borate
- 0.002 M EDTA
Procedure
DBD-FISH for detection and quantification of X-ray induced initial DNA
breakage (Fernandez et al. 1998).
Microgel on slide 1. 1% low melting point agarose in water is kept at 37°C in a water bath.
2. Isolated cells in suspension (e.g. buffy coat or cells in culture medium)
are gently mixed with the agarose to achieve the latter at a final concentration of 0.7%, at 37°C.
3. 50 Jll of the suspension are pipetted onto a glass slide coated with a thin
dry film of 0.65% standard agarose, and immediately covered with a
glass coverslip (24 x 60 mm). Coating has been performed by immersing vertically for 2s the glass slide in the liquid standard agarose solution. After solidifying horizontally on a cold plate, it was dried at 80°C.
Other systems such as "sandwich" gels on frosted slides (Fairbain et aL
1995) can also be applied.
JosE LUIS FERNANDEZ, VICENTE GOYANES, and JAIME GOSALVEZ
- 0.03 M NaOH
- 1 M NaCl
- Neutralising and lysing solution 1 (pH 7.5), stored at 4°C, used at room
temperature
- 0.4 M Tris HCl
- 0.8 M DTT (Irritant)
- 1% SDS (Harmful if swallowed. Irritant)
- 0.05 M EDTA
- Neutralising and lysing solution 2 (pH 7.5), stored at 4°C, used at room
temperature
- 0.4 M Tris HCl
- 2 M NaCl
- 1% SDS
- Neutralising solution (pH 7.5), stored at 4°C, used at room temperature: 0.4 M Tris HCl
- Isotonic saline solution, stored at 4°C: 0.9% NaCl
- TBE buffer (pH 7.5), stored at room temperature
- 0.09 M Tris borate
- 0.002 M EDTA
Procedure
DBD-FISH for detection and quantification of X-ray induced initial DNA
breakage (Fernandez et al. 1998).
Microgel on slide 1. 1% low melting point agarose in water is kept at 37°C in a water bath.
2. Isolated cells in suspension (e.g. buffy coat or cells in culture medium)
are gently mixed with the agarose to achieve the latter at a final concentration of 0.7%, at 37°C.
3. 50 Jll of the suspension are pipetted onto a glass slide coated with a thin
dry film of 0.65% standard agarose, and immediately covered with a
glass coverslip (24 x 60 mm). Coating has been performed by immersing vertically for 2s the glass slide in the liquid standard agarose solution. After solidifying horizontally on a cold plate, it was dried at 80°C.
Other systems such as "sandwich" gels on frosted slides (Fairbain et aL
1995) can also be applied.