Breast cancer early warning chip for easily rapid measuring human I type thymidine kinase gene protein
A technology for rapid determination of thymidine kinase, applied in measuring devices, biological testing, material inspection products, etc., can solve the problems of unsuitable large-scale screening, difficult application, low sensitivity, etc.
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[0052] ① Cloning and sequence analysis of hTK1 gene:
[0053] 1) Amplify TK1 cDNA by reverse transcription PCR: Extract RNA from 5 million Hela cells with Trizol kit, which can extract total ribonucleic acid (RNA) from animal cells and tissues, bacteria, fungi, etc., and treat with methotrexate 48 After purification, the precipitate was treated with a high-efficiency alkylating agent-diethyl pyrocarbonate (DEPC) to dissolve the precipitate in water to obtain 30 μl. Since DEPC can destroy ribonuclease (RNase) activity, it can remove RNase contamination.
[0054] When performing reverse transcription, add 5-fold buffer: 4 μl, primer Oligo-dT (0.5 μg / 1 μl) 1 μl, deoxynucleoside triphosphate (dNTP) at 10 mMol / L concentration: 2 μl, RNA: 10 μl, deionized water : 2 μl, mixed thoroughly, and placed in a constant temperature water bath at 70°C for 10 minutes; then, placed on ice for 2 minutes; added 1 μl reverse transcriptase (50 U, Roche), mixed well, and placed in a water bath at 4...
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