266
JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
- Microscope slides and coverslips
- Hoechst 33258
- Ultraviolet light source, long-wave (-365 nM, see comment I)
- Exonuclease III with buffer. Most companies supply the appropriate
buffer with the enzyme, although any buffer recommended for use
with exonuclease III can be used.
- Labeled, single-stranded DNA probe (see comment 2)
- Formamide
- 20xSSC, pH 7, stock solution (l xSSC = 0.15M sodium chloride and
0.015 M sodium citrate)
- 2xSSC solution prepared from 20xSSC stock solution
- PN Buffer made by titrating O.IM solutions of dibasic and monobasic
sodium phosphate to give a solution at pH 8, then add Triton X-100 to
final concentration of 1%
- Fluorescent counterstain, such as DAPI
- Antifade solution
- Fluorescence microscope with camera and/or image analysis system
Procedure
Bromodeoxyuridine incorporation
Any type of cell culture with a reasonable rate of growth can be used for
CO-FISH. It is desirable to know the approximate doubling time of the
cells to determine the optimal time required for most of the cells to complete one round of DNA replication.
1. Add bromodeoxyuridine stock solution to cell culture medium to
achieve a final concentration of 105
M. BrdU concentration may
need to be adjusted for some cell cultures. Other nucleotide analogues
may also be used but results may vary.
2. Incubate the cell culture for a period of time equal to about one doubling time of the cells being used.
JULIANNE MEYNE, SUSAN M. BAILEY, and EDWIN H. GOODWIN
- Microscope slides and coverslips
- Hoechst 33258
- Ultraviolet light source, long-wave (-365 nM, see comment I)
- Exonuclease III with buffer. Most companies supply the appropriate
buffer with the enzyme, although any buffer recommended for use
with exonuclease III can be used.
- Labeled, single-stranded DNA probe (see comment 2)
- Formamide
- 20xSSC, pH 7, stock solution (l xSSC = 0.15M sodium chloride and
0.015 M sodium citrate)
- 2xSSC solution prepared from 20xSSC stock solution
- PN Buffer made by titrating O.IM solutions of dibasic and monobasic
sodium phosphate to give a solution at pH 8, then add Triton X-100 to
final concentration of 1%
- Fluorescent counterstain, such as DAPI
- Antifade solution
- Fluorescence microscope with camera and/or image analysis system
Procedure
Bromodeoxyuridine incorporation
Any type of cell culture with a reasonable rate of growth can be used for
CO-FISH. It is desirable to know the approximate doubling time of the
cells to determine the optimal time required for most of the cells to complete one round of DNA replication.
1. Add bromodeoxyuridine stock solution to cell culture medium to
achieve a final concentration of 105
M. BrdU concentration may
need to be adjusted for some cell cultures. Other nucleotide analogues
may also be used but results may vary.
2. Incubate the cell culture for a period of time equal to about one doubling time of the cells being used.