Klenow Fragment (Large Fragment of DNA Polymerase I):
[40 U/ul Klenow Fragment in 50 mM Potassium Phosphate (pH 7.0), 100 mM KCl,1 mM DTT, 50% Glycerol]
Stop Buffer: [0.5 M Na2EDTA (pH 8.0)]
Distilled Water
Procedures:
1. Dissolve 100 ng DNA in 5-20 ul of dilute buffer in a microcentrifuge tube. On ice, add 20 μl 2.5X Random Primers Solution.
2. Denature by heating for 5 min in a boiling water bath; immediately cool on ice. (The amount of template per reaction has been varied from 25-500 ng with satisfactory results.)
Perform the following additions on ice:
5 ul 10X dNTP Mixture
Distilled Water to a total volume of 49 ul
3. Mix briefly.
4. Add 1 ul Klenow Fragment. Mix gently but thoroughly. Centrifuge 15-30 sec.
5. Incubate at 37℃ for 60 min.
6. Add 5 ul Stop Buffer.
Probes Labeling for FISH by DOP-PCR: (see Protocol of Chromosome Microdissection)
vHybridization:
1. Materials:1.1. Slide Pretreatment 1 (Optional: not recommended for metaphase FISH): PK stock solution: 5 mg proteinase K (Boehringer, Mannheim, Germany), 50 mL 1M Tris-HCl (pH 7.5), 20 mL 0.5M EDTA (pH 7.0), 2 mL 5M NaCl, make up to 1 mL in filtered double- distilled water; make fresh as required.
2.20X standard saline citrate (SSC) stock solution: 3.0M NaCl, 0.3M Na-citrate; set up with double-distilled water, adjust to pH 7.0, autoclave, and store at room temperature.
3.RNase stock solution: 10 mg/mL of RNase type A (Boehringer); set up with filtered double- distilled water; aliquot and store at -20ºC.
4.RNase solution: per slide 200 mL 2X SSC plus 10 mL of RNase stock solution are necessary; make fresh as required.
5. (Optional: not recommended for metaphase FISH): Pepsin stock solution 10% (w/v): dissolve 100 mg pepsin (Serva, Heidelberg, Germany) in 1 mL of filtered double-distilled water at 37ºC; aliquot and store at -20ºC.
6. (Optional: not recommended for metaphase FISH): Pepsin buffer: Add 1 mL of 1M HCl to 99 mL of distilled water and incubate at 37ºC for about 20 min; then add 50 mL of the pepsin stock solution 10% (wlv) and leave the coplin jar at 37ºC; make fresh as required.
7.(Optional: not recommended for metaphase FISH): 1X PBS/ MgCl2: 5% (v/v) 1M MgCl2 in 1X PBS. (2.5mL 1M MgCl2 in 47.5mL 1X PBS)
8.(Optional: not recommended for metaphase FISH): Formalin buffer: 3% (v/v) of acid-free formaldehyde (37%; Roth) in 1X PBS; make fresh as required.
