Loss of Heterozygosity
This method was successful in our lab using prostate tissue and for our specific objectives. Investigators must be aware that they will need to tailor the following protocol for their own research objectives and tissue under study. This method is used to detect genomic DNA deletions in tumor cells. For a more detailed discussion of applying this approach to microdissected samples, see Allelic Loss Studies in Prostate MP at NCI. 1. Reagents DNA sample (see Processing of Microdissected Tissue - DNA-based Analysis) Proteinase K (Sigma) Proteinase K buffer (0.05 M tris-HCL, 0.001 M EDTA, 1% Tween 20, 0.1 mg/ml proteinase K, pH 8.0) Ampli Taq Gold Buffer (Perkin Elmer) dNTP mixture (Perkin Elmer) Primers DEPC-treated H2O Ampli Taq Gold Polymerase (Perkin Elmer) a-32P dCTP, 6000 Ci/mmol (NEN Dupont) Formamide, 99% (Fluka) Bromophenol blue-Xylene cyanole (Sigma), reconstituted as directed Gel Mix-6 sequencing gel solution (Life Technologies) Ammonium persulfate (Biorad) 10X TBE buffer (0.89 M Tris Base, 0.89 M Boric Acid 0.02M Disodium EDTA ) (Advanced Biotechnologies) Acrylease (Stratagene) Glass cleaner (e.g., Windex, Glass Plus) 95% ethanol 2. Equipment Thermal cycler (MJ Research) Sequencing gel electrophoresis apparatus (Gibco BRL) High voltage power supply Gel dryer (Life Technologies) Glass plates, 31.0 x 38.5 cm (Life Technologies) 0.4 mm spacers (Life Technologies) Casting boot (Life Technologies) Shark tooth comb (Life Technologies) Small clamps Whatman blotting paper, 3 mm thickness Kodak Biomax MR or AR film Film cassette (Amersham Life Science) Film processor [1][2][3][4][5]下一页
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