Method of detecting apolipoprotein E gene type and kit
A technology of apolipoprotein and genotype, which is applied in the direction of biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of high cost, insufficient resolution, complicated technology, etc., and achieve good detection stability and accurate and reliable results Effect
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[0079] Example 1: Preparation of sample target nucleic acid
[0080] Use a sterile syringe to draw 1ml of the subject's venous blood, add it to an anticoagulation tube containing EDTA, and store at room temperature or low temperature. Take 300μl of anticoagulated whole blood and add it to a 1.5ml centrifuge tube, add 1ml of sterilized pure water to the centrifuge tube, mix well, and let it stand at room temperature for 2-4 minutes. It was then centrifuged (5000 rpm) for 6 minutes, and the precipitate was collected. Repeat the above steps once, add 1ml of physiological saline to the centrifuge tube and mix it, centrifuge at 10000rpm for 10 minutes, collect the white precipitate at the bottom of the tube. Add 50 μl of DNA extract to the pellet, mix well, boil the water for 10 minutes, and centrifuge at 12000 rpm for 10 minutes. Take 2μl of supernatant as PCR reaction template.
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[0081] Example 2: PCR amplification of target nucleic acid
[0082] Take a single tube of single PCR reaction solution, add 1μl UDG enzyme to each tube, then directly add 2μl template (or negative and positive standard), mix well, centrifuge briefly (3 seconds), and put each reaction tube into PCR After pretreatment at 50°C for 3 minutes, hot-start combined with "landing PCR" was used to amplify according to the following conditions: 94°C for 4 minutes, 80°C for 3 minutes, and 1μl of Taq enzyme was added in the process. Denaturation at 94°C for 2 minutes, then press 94°C for 1 minute, 70°C for 1 minute, 72°C for 1 minute, a total of 5 cycles, and then press 94°C for 1 minute, 64°C for 1 minute, and 72°C for 1 minute, for a total of 30 cycles. Finally, it was extended at 72°C for 7 minutes. The amplified products were detected by 2% agarose gel electrophoresis (see attached figure 2 ).
Example Embodiment
[0083] Example 3: Reverse dot blot detection of samples with six known genotypes
[0084] Before hybridization, take hybridization solution I (2×SSC-0.1% SDS) and mix it with hybridization solution II and preheat to 59°C for use. Take 6 1.5ml centrifuge tubes according to the number of samples to be tested, add 0.5ml hybridization solution I to each tube and preheat to 59°C. After denaturing the PCR amplified product at 97°C for 5 minutes, place it in an ice-water mixture for 2-5 minutes. Then take 1000μl hybridization solution I+2μl solution I (1000: 2) mixed solution as the binding solution and store at 4°C for later use, and take the mixture of solution II: solution III: solution IV (1900: 200:1) (19ml solution II+2ml) Solution III+10μl solution IV) was used as a color developing solution and protected from light for later use.
[0085] Fill the reaction chamber with distilled water before hybridization, place the metal perforated plate, and turn on the water pump to drain the ...
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