Kit for detecting SNP (Single Nucleotide Polymorphism) sites related to Warfarin individualized application, and multiplex PCR (Polymerase Chain Reaction) amplification method and detection method using same
A kit and site technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of decreased arachidonic acid metabolism, decreased CYP4F2 enzymatic activity, decreased arachidonic acid production, etc. , to achieve the effect of reducing detection cost and operation complexity, reducing operation steps and avoiding aerosol pollution
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[0067] Example 1
[0068] Step 1: Preparation of Whole Blood Cell Lysate
[0069] Take 300 μl of peripheral venous blood of the subject, add 700 μl of cell lysate, invert and mix 5 times, centrifuge at 12,000 rpm for 1 minute, discard the supernatant, and place the centrifuge tube upside down on clean absorbent paper for 2 minutes to ensure that the pellet is in the tube , add 300 μl of double-distilled water, and mix by vortexing to form a cell lysis suspension.
[0070] Step 2: PCR Amplification Reaction
[0071] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer and 0.2 μl of polymerase (2.5U / μl) into the PCR tube to form an independent reaction system. Homogenize, centrifuge briefly, and finally gently add 20 μl of liquid paraffin oil along the tube wall to seal the system, as shown in the following table:
[0072] 2x wild-type amplification buffer
[0073] 2. Configure the mutant reaction...
Example Embodiment
[0087] Embodiment 2
[0088] Step 1: Extraction and Dilution of Whole Blood Genomic DNA
[0089] Take 300 μl of peripheral venous blood of the subject, and extract whole blood genomic DNA according to the instructions of the whole blood genomic DNA extraction kit. Measure the concentration of DNA with a spectrophotometer and dilute to 15-20 ng / μl.
[0090] Step 2: PCR Amplification Reaction
[0091] 1. Configure the wild-type reaction system: add 4.8 μl of cell lysis suspension, 5 μl of 2× wild-type amplification buffer and 0.2 μl of polymerase (2.5U / μl) into the PCR tube to form an independent reaction system. Homogenize, centrifuge briefly, and finally gently add 20 μl of liquid paraffin oil along the tube wall to seal the system, as shown in the following table:
[0092] 2x wild-type amplification buffer
[0093] 2. Configure the mutant reaction system: add 4.8 μl of cell lysis suspension, 5 μl 2× mutant amplification buffer and 0.2 polymerase (2.5 U / μl) to the P...
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