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YOSHITAKA HISHIKAWA ET AL.
Dot blot hybridization
Day 1 1. Prepare strips of nitrocellulose filters.
2. Denature DNA by boiling for 7 mins.
Note: Use of DNA dissolved in DDW alone or excessively prolonged incubation will result in fragmentation of the DNA.
3. Apply 2 III of DNA solution containing DNA at various quantities (10 ng,l ng,100 pg, 10 pg, 1 pg).
Note: Be sure not to use the T-T dimered cDNA at this time.
4. Dry the filter completely (overnight air-dry is recommended).
Day 2 5. Bake the filter at Soac for 2 hrs.
6. Prehybridization. Prehybridize the filter for 2 hrs at 42°C inside a vinyl bag (250 IlI)(see Table 1. Prehybridization solution).
Table 1. Prehybridization solution
Hybridization mediums
lOmM Tris/HCI (pH 7.4)
0.6M NaCl
ImM EDTA
40% Deionized formamide
Ix Denhardt's solution
500~g/ml yeast tRNA
Reagents
volume (~l)
1M Tris/HCl (pH 7.4)
2.5
5M NaCl
30
200mM EDT A
1.25
Deionized formamide
100
100x Denhardt's solution
2.5
lOmg/ml yeast tRNA
12.5
250~g/ml salmon sperm DNA lOmg/ml salmon
sperm DNA
6.25
Sterile DDW
total (for 1 bag)
Note. The total mixture should be boiled for 7 mins.
Note. Use 250 ~l for 1 filter in one vinyl bag.
95
250