Method Article
A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.
A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH. Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
Solution | Reagents | Comments | Storage |
B Cell Wash Buffer | 500 ml 1x Hanks Balanced Salt Solution | Filter through 0.22 μm stericup filtration unit | 4 °C |
5 ml 50 °C heat inactivated FCS | |||
5 ml 100x Pen/Strep | |||
B Cell Medium | 418.5 ml RPMI-1640 | Filter through 0.22 μm stericup filtration unit | 4 °C |
50 ml 50 °C heat inactivated FCS | |||
5 ml 100x Pen/Strep | |||
5 ml glutamine | |||
5 ml nonessential amino acids | |||
5 ml sodium pyruvate | |||
1.8 ml 14.2 M 2-mercaptoethanol | |||
5 ml 1M HEPES | |||
Fixative | 3:1 v/v methanol/glacial acetic acid | Make fresh | |
0.075 M KCl | 1.395 g KCl | Room Temperature | |
250 ml distilled H2O | |||
FISH Master Mix | 40 ml 50% Dextran sulfate | Vortex the solution and leave in a shaking platform overnight | Aliquot and store at -20 °C |
20 ml 20x SSC | |||
40 ml sterile distilled H2O | |||
70% Formamide/2x SSC | 10 ml 20x SSC | Adjust to pH 7 with 1M HCl | Aliquot and store at -20 °C |
20 ml distilled H2O | |||
70 ml deionized formamide | |||
0.01 M HCl | 1 ml 1M HCl | Adjust to pH 2.0 | Room Temperature |
99 ml distilled H2O | |||
1x PBS/MgCl2 | 50 ml 1M MgCl2 | Room Temperature | |
950 ml 1x PBS | |||
50% Formamide/2x SSC | 20 ml 20x SSC | Adjust to pH 7.25 Formamide used in this step does not have to be deionized. | Make fresh |
80 ml distilled H2O | |||
100 ml formamide | |||
4x SSC/0.1% Tween20 | 50 ml 20x SSC | Room Temperature | |
200 ml distilled H2O | |||
250 μl Tween20 | |||
DAPI staining solution (80 ng/ml) | 40 μl 0.2mg/ml DAPI stock | 4 °C in light protected staining jar | |
100 ml 2x SSC | |||
Mowiol Mounting Medium | 6 g glycerol | Incubate at 50 °C with stirring for 2hrs | Aliquot and store at 4 °C |
2.4 g Mowiol 4-88 | |||
6 ml distilled H2O | |||
12 ml 0.2 M TrisCl pH 8.5 | |||
230 μl 1% Thimerosal (w/v in water) |
Table 1. List of solutions.
A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH. Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
Solution | Reagents | Comments | Storage |
B Cell Wash Buffer | 500 ml 1x Hanks Balanced Salt Solution | Filter through 0.22 μm stericup filtration unit | 4 °C |
5 ml 50 °C heat inactivated FCS | |||
5 ml 100x Pen/Strep | |||
B Cell Medium | 418.5 ml RPMI-1640 | Filter through 0.22 μm stericup filtration unit | 4 °C |
50 ml 50 °C heat inactivated FCS | |||
5 ml 100x Pen/Strep | |||
5 ml glutamine | |||
5 ml nonessential amino acids | |||
5 ml sodium pyruvate | |||
1.8 ml 14.2 M 2-mercaptoethanol | |||
5 ml 1M HEPES | |||
Fixative | 3:1 v/v methanol/glacial acetic acid | Make fresh | |
0.075 M KCl | 1.395 g KCl | Room Temperature | |
250 ml distilled H2O | |||
FISH Master Mix | 40 ml 50% Dextran sulfate | Vortex the solution and leave in a shaking platform overnight | Aliquot and store at -20 °C |
20 ml 20x SSC | |||
40 ml sterile distilled H2O | |||
70% Formamide/2x SSC | 10 ml 20x SSC | Adjust to pH 7 with 1M HCl | Aliquot and store at -20 °C |
20 ml distilled H2O | |||
70 ml deionized formamide | |||
0.01 M HCl | 1 ml 1M HCl | Adjust to pH 2.0 | Room Temperature |
99 ml distilled H2O | |||
1x PBS/MgCl2 | 50 ml 1M MgCl2 | Room Temperature | |
950 ml 1x PBS | |||
50% Formamide/2x SSC | 20 ml 20x SSC | Adjust to pH 7.25 Formamide used in this step does not have to be deionized. | Make fresh |
80 ml distilled H2O | |||
100 ml formamide | |||
4x SSC/0.1% Tween20 | 50 ml 20x SSC | Room Temperature | |
200 ml distilled H2O | |||
250 μl Tween20 | |||
DAPI staining solution (80 ng/ml) | 40 μl 0.2mg/ml DAPI stock | 4 °C in light protected staining jar | |
100 ml 2x SSC | |||
Mowiol Mounting Medium | 6 g glycerol | Incubate at 50 °C with stirring for 2hrs | Aliquot and store at 4 °C |
2.4 g Mowiol 4-88 | |||
6 ml distilled H2O | |||
12 ml 0.2 M TrisCl pH 8.5 | |||
230 μl 1% Thimerosal (w/v in water) |
Table 1. List of solutions.
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